JP7039694B2 - Anti-APOC3 antibody and how to use it - Google Patents

Description

Related Applications This application claims the interests of US Provisional Patent Application No. 62 / 579,449 filed October 31, 2017, which is incorporated herein by reference in its entirety.

The present disclosure relates to antibodies that specifically bind to ApoC3 (eg, human ApoC3) and the methods thereof used.

Elevated blood triglyceridemia levels (hypertriglyceridemia) are causative factors of atherosclerosis and increase the risk of cardiovascular events such as cardiovascular death, angina, myocardial infarction, and stroke.

ApoC3 is a protein that circulates in the blood at very high concentrations (> 10 μM), most of which bind to triglyceride-rich lipoproteins (TRLs), TRL remnants, and high-density lipoproteins. ApoC3 appears to be an important regulator of blood triglyceride levels. For example, ApoC3 levels in humans have been shown to be positively correlated with blood triglyceride levels, and elevated ApoC3 levels are associated with hypertriglyceridemia. In addition, ApoC3 inhibits the activity of lipoprotein lipase, an enzyme that hydrolyzes TRL triglycerides, and also inhibits the uptake of TRL remnants into the liver, both of which cause elevated blood triglyceride levels.

WO2014 / 131008 WO 2018/193427 WO2018 / 007999

Several treatments have been approved for the treatment of hypertriglyceridemia such as fibrates, niacin, and omega-3 fatty acids. However, these therapies are only somewhat effective in reducing plasma triglycerides. Therefore, there is a need for improved treatments in the art to reduce plasma triglycerides.

The present disclosure provides antibodies that specifically bind to ApoC3 (eg, human ApoC3) and inhibit ApoC3 function. Pharmaceutical compositions containing these antibodies, nucleic acids encoding these antibodies, expression vectors and host cells for making these antibodies, and methods of treating a subject with these antibodies are also provided.

In certain embodiments, the anti-ApoC3 antibodies disclosed herein can diminish the ability of ApoC3 to inhibit the uptake of TRL by hepatocytes, and when administered to a subject, the serum levels of ApoC3 and ApoB. It can be reduced rapidly and persistently. Therefore, the disclosed anti-ApoC3 antibodies are useful in the treatment and prevention of hypertriglyceridemia and related diseases (eg, cardiovascular disease and pancreatitis).
Thus, in one aspect, the disclosure provides an isolated antibody that specifically binds to _ApoC3 at pH 7.4 with a first dissociation constant (KD ₎ and at pH 5.5 with a second KD. , The ratio of the second _KD to the first _KD is at least about 5, 10, 20, or 50. In certain embodiments, the first _KD is less than 10, 5, 2, 1, 0.5, 0.2, or 0.1 nM. In certain embodiments, the half-life of an antibody in mice expressing ApoC3 is at least about 3, 7, 14, 21, or 28 days.

In certain embodiments, the antibody diminishes the ability of ApoC3 to inhibit hepatocyte uptake of very low density lipoprotein (VLDL). In certain embodiments, the antibody is capable of increasing the clearance rate of ApoC3 from the blood of interest. In certain embodiments, the antibody can increase the clearance rate of ApoB from the blood of interest. In certain embodiments, the antibody can reduce the level of ApoC3 in the blood of the subject. In certain embodiments, the antibody can reduce the level of ApoC3 in the blood of a subject by at least 40% for at least 2 weeks. In certain embodiments, the antibody can reduce the level of ApoB in the blood of the subject. In certain embodiments, the antibody can reduce the level of ApoB in the blood of a subject by at least 20% for at least 2 weeks. In certain embodiments, the antibody is capable of inhibiting postprandial lipoemia in a subject. In certain embodiments, the antibody is capable of binding to the lipid binding ApoC3.

In certain embodiments, the antibody binds to an epitope within the amino acid sequence set forth in SEQ ID NO: 2. In certain embodiments, the epitope comprises at least one amino acid at position 2, 5, 6, 8, or 10 of SEQ ID NO: 2. In certain embodiments, the epitope comprises an amino acid at positions 5 and 6 of SEQ ID NO: 2. In certain embodiments, the epitope comprises an amino acid at positions 2, 5, 6, and 8 of SEQ ID NO: 2. In certain embodiments, the epitope comprises an amino acid at position 10 of SEQ ID NO: 2. In certain embodiments, the epitope comprises an amino acid at positions 6, 8, and 10 of SEQ ID NO: 2. In certain embodiments, the epitope comprises an amino acid at positions 6 and 8 of SEQ ID NO: 2.

In certain embodiments, the antibody (eg, a humanized antibody) has a heavy chain variable region having complementarity determining regions CDRH1, CDRH2, and CDRH3 and a light chain variable region having complementarity determining regions CDRL1, CDRL2, and CDRL3. And, including
(A) CDRH1 comprises the amino acid sequence of TYSMR (SEQ ID NO: 3).
(B) CDRH 2 comprises the amino acid sequence of SIHTX ₁ X ₂ GGTAYRDSVKG (where X ₁ is G, E, or D and X ₂ is G or A) (SEQ ID NO: 87).
(C) CDRH3 comprises the amino acid sequence of AGYSD (SEQ ID NO: 10).
(D) CDRL1 comprises the amino acid sequence of KTSQGLVHSXGKTYFY (where X is D or G in the formula) (SEQ ID NO: 88).
(E) CDRL2 comprises the amino acid sequence of QVSNRAS (SEQ ID NO: 7), and (f) CDRL3 comprises the amino acid sequence of AXGTYYPHT (where X is Q or H in the formula) (SEQ ID NO: 8). Optionally, the antibodies CDRH1, CDRH2, and CDRH3 are not SEQ ID NOs: 3, 9, 10; 3, 11, 10; 3, 9, 12; or 3, 11, 12, respectively.

In certain embodiments, CDRH2 comprises the amino acid sequence of SIHTGGGTAYRDSVKG (SEQ ID NO: 36), SIHTAGEGGTAYRDSVKG (SEQ ID NO: 37), SIHTDAGGTAYRDSVKG (SEQ ID NO: 38), or SIHTGGGTAYRDSVKG (SEQ ID NO: 39). In certain embodiments, CDRL1 comprises the amino acid sequence of KTSQGLVHSDGKTYFY (SEQ ID NO: 6) or KTSQGLVHSGGKTYFY (SEQ ID NO: 40). In certain embodiments, CDRL3 comprises the amino acid sequence of AHGTYYPHT (SEQ ID NO: 14) or AQGTYYPHT (SEQ ID NO: 13).

In certain embodiments, CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3 are SEQ ID NOs: 3, 36, 10, 6, 7, and 14; 3, 37, 10, 40, 7, and 14, respectively; 3, 38, 10, 40, 7, and 14; 3, 38, 10, 6, 7, and 14; 3, 39, 10, 6, 7, and 14; or 3, 37, 10, 40, 7, And the amino acid sequence shown in 13. In certain embodiments, the heavy chain variable region comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 42-53. In certain embodiments, the light chain variable region comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 54-65. In certain embodiments, the heavy chain variable region and the light chain variable region are represented by SEQ ID NOs: 42 and 54, 43 and 55, 44 and 56, 45 and 57, 46 and 58, 46 and 54, 47 and 58, 47, respectively. And 54, 48 and 58, 48 and 54, 49 and 59, 49 and 60, 50 and 59, 50 and 60, 51 and 61, 52 and 62, 53 and 62, 43 and 63, 44 and 64, or 45 and Contains the amino acid sequence shown in 65.

In certain embodiments, the antibody comprises heavy chain variable regions having complementarity determining regions CDRH1, CDRH2, and CDRH3 and light chain variable regions having complementarity determining regions CDRL1, CDRL2, and CDRL3.
(A) CDRH1 comprises the amino acid sequence of TYSMR (SEQ ID NO: 3).
(B) CDRH 2 comprises the amino acid sequence of SIX ₁ TDGGGTAYRDSVKG (where X ₁ is S or H in the formula) (SEQ ID NO: 4).
(C) CDRH3 comprises the amino acid sequence of X ₂ GYSD (where X ₂ is A or H in the formula) (SEQ ID NO: 5).
(D) CDRL1 comprises the amino acid sequence of KTSQGLVHSDGKTYFY (SEQ ID NO: 6).
(E) CDRL2 comprises the amino acid sequence of QVSNRAS (SEQ ID NO: 7), and (f) CDRL3 is the amino acid sequence of AX _{3 GTYYPHT} (where X3 is Q or H in the formula) (SEQ ID NO: 8). Including
In the formula, at least one of X ₁ , X ₂ , and X ₃ is H.

In certain embodiments, CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3 are SEQ ID NOs: 3, 11, 10, 6, 7, and 13; 3, 9, 12, 6, 7, and 13; 3, 9, 10, 6, 7, and 14; 3, 11, 10, 6, 7, and 14; 3, 9, 12, 6, 7, and 14; 3, 11, 12, 6, 7, and 13; Or it comprises the amino acid sequences shown in 3, 11, 12, 6, 7, and 13. In certain embodiments, the heavy chain variable region comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 16-18. In certain embodiments, the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 20. In certain embodiments, the heavy and light chain variable regions are assigned to SEQ ID NOs: 16 and 19, 17 and 19, 18 and 19, 15 and 20, 16 and 20, 17 and 20, or 18 and 20, respectively. Contains the amino acid sequence shown.

In another aspect, the disclosure comprises a heavy chain variable region having complementarity determining regions CDRH1, CDRH2, and CDRH3 and a light chain variable region having complementarity determining regions CDRL1, CDRL2, and CDRL3 in ApoC3. Provided isolated antibodies that specifically bind (eg, humanized antibodies),
(A) CDRH1 comprises the amino acid sequence of TYSMR (SEQ ID NO: 3).
(B) CDRH 2 comprises the amino acid sequence of SIHTX ₁ X ₂ GGTAYRDSVKG (where X ₁ is G, E, or D and X ₂ is G or A) (SEQ ID NO: 87).
(C) CDRH3 comprises the amino acid sequence of AGYSD (SEQ ID NO: 10).
(D) CDRL1 comprises the amino acid sequence of KTSQGLVHSXGKTYFY (where X is D or G in the formula) (SEQ ID NO: 88).
(E) CDRL2 comprises the amino acid sequence of QVSNRAS (SEQ ID NO: 7), and (f) CDRL3 comprises the amino acid sequence of AXGTYYPHT (where X is Q or H in the formula) (SEQ ID NO: 8).

In certain embodiments, CDRH2 comprises the amino acid sequence of SIHTGGGTAYRDSVKG (SEQ ID NO: 36), SIHTAGEGGTAYRDSVKG (SEQ ID NO: 37), SIHTDAGGTAYRDSVKG (SEQ ID NO: 38), or SIHTGGGTAYRDSVKG (SEQ ID NO: 39). In certain embodiments, CDRL1 comprises the amino acid sequence of KTSQGLVHSDGKTYFY (SEQ ID NO: 6) or KTSQGLVHSGGKTYFY (SEQ ID NO: 40). In certain embodiments, CDRL3 comprises the amino acid sequence of AHGTYYPHT (SEQ ID NO: 14) or AQGTYYPHT (SEQ ID NO: 13). In certain embodiments, CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3 are SEQ ID NOs: 3, 36, 10, 6, 7, and 14; 3, 37, 10, 40, 7, and 14, respectively; 3, 38, 10, 40, 7, and 14; 3, 38, 10, 6, 7, and 14; 3, 39, 10, 6, 7, and 14; or 3, 37, 10, 40, 7, And the amino acid sequence shown in 13.

In another aspect, the disclosure provides an isolated antibody that specifically binds to ApoC3 (eg, a humanized antibody), wherein the antibody comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 42-53. Contains heavy chain variable regions.

In another aspect, the disclosure provides an isolated antibody that specifically binds to ApoC3 (eg, a humanized antibody), wherein the antibody comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 54-65. Contains the light chain variable region containing.

In another aspect, the disclosure provides an isolated antibody that specifically binds to ApoC3 (eg, a humanized antibody), wherein the antibody comprises a heavy chain variable region and a light chain variable region and is heavy chain variable. The regions and light chain variable regions are SEQ ID NOs: 42 and 54, 43 and 55, 44 and 56, 45 and 57, 46 and 58, 46 and 54, 47 and 58, 47 and 54, 48 and 58, 48 and, respectively. Includes the amino acid sequences set forth in 54, 49 and 59, 49 and 60, 50 and 59, 50 and 60, 51 and 61, 52 and 62, 53 and 62, 43 and 63, 44 and 64, or 45 and 65.

In another aspect, the disclosure comprises a heavy chain variable region having complementarity determining regions CDRH1, CDRH2, and CDRH3 and a light chain variable region having complementarity determining regions CDRL1, CDRL2, and CDRL3 in ApoC3. Provided isolated antibodies that specifically bind,
(A) CDRH1 comprises the amino acid sequence of TYSMR (SEQ ID NO: 3).
(B) CDRH 2 comprises the amino acid sequence of SIX ₁ TDGGGTAYRDSVKG (where X ₁ is S or H in the formula) (SEQ ID NO: 4).
(C) CDRH3 comprises the amino acid sequence of X ₂ GYSD (where X ₂ is A or H in the formula) (SEQ ID NO: 5).
(D) CDRL1 comprises the amino acid sequence of KTSQGLVHSDGKTYFY (SEQ ID NO: 6).
(E) CDRL2 comprises the amino acid sequence of QVSNRAS (SEQ ID NO: 7), and (f) CDRL3 is the amino acid sequence of AX _{3 GTYYPHT} (where X3 is Q or H in the formula) (SEQ ID NO: 8). Including
In the formula, at least one of X ₁ , X ₂ , and X ₃ is H.

In certain embodiments, CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3 are SEQ ID NOs: 3, 11, 10, 6, 7, and 13; 3, 9, 12, 6, 7, and 13; 3, 9, 10, 6, 7, and 14; 3, 11, 10, 6, 7, and 14; 3, 9, 12, 6, 7, and 14; 3, 11, 12, 6, 7, and 13; Or it comprises the amino acid sequences shown in 3, 11, 12, 6, 7, and 13.

In another aspect, the disclosure provides an isolated antibody that specifically binds to ApoC3, wherein the antibody comprises a heavy chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 16-18. ..

In another aspect, the disclosure provides an isolated antibody that specifically binds to ApoC3, wherein the antibody comprises a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 20.

In another aspect, the present disclosure provides an isolated antibody that specifically binds to ApoC3, wherein the antibody comprises a heavy chain variable region and a light chain variable region, the heavy chain variable region and the light chain variable region. , Each comprising the amino acid sequences set forth in SEQ ID NOs: 16 and 19, 17 and 19, 18 and 19, 15 and 20, 16 and 20, 17 and 20, or 18 and 20, respectively.

In a particular embodiment of any one of the aforementioned embodiments, the antibody further comprises a human or humanized constant region. In certain embodiments, the constant region is a variant of the wild human immunoglobulin heavy chain constant region, and the variant human immunoglobulin heavy chain constant region is the affinity of the wild human immunoglobulin heavy chain constant region for human FcRn at pH 6. Compared to sex, it has an increased affinity for human neonatal Fc receptors (FcRn) at pH 6. In certain embodiments, the constant region is the heavy chain constant region of human IgG. In certain embodiments, the constant region is a heavy chain constant region of human IgG ₁ , IgG ₂ , or IgG ₄ .

In certain embodiments, the constant region comprises amino acids K, F, and Y at EU positions 433, 434, and 436, respectively. In certain embodiments, the constant region comprises the amino acids Y, T, and E at EU positions 252, 254, and 256, respectively. In certain embodiments, the constant region comprises amino acids L and S at EU positions 428 and 434, respectively. In certain embodiments, the constant region comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 22-24, 76-78, and 81-86.

In certain embodiments, the isolated antibody comprises a heavy chain comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 66-73. In certain embodiments, the isolated antibody comprises a light chain comprising the amino acid sequence set forth in SEQ ID NO: 74. In certain embodiments, the isolated antibody comprises a heavy chain and a light chain, wherein the heavy and light chains are SEQ ID NOs: 66 and 74, 67 and 74, 68 and 74, 69 and 74, 70 and, respectively. Includes the amino acid sequences set forth in 74, 71 and 74, 72 and 74, or 73 and 74.

In another aspect, the disclosure provides a pharmaceutical composition comprising the antibodies disclosed herein and a pharmaceutically acceptable carrier.

In another aspect, the disclosure provides a polynucleotide encoding a heavy or light chain variable region of an antibody disclosed herein. In another aspect, the disclosure provides an expression vector comprising the polynucleotides disclosed herein. In another aspect, the disclosure provides a host cell comprising the expression vector disclosed herein.

In another aspect, the disclosure provides a method for producing an antibody that binds to ApoC3, wherein the host cells disclosed herein are cultured under conditions that allow expression of the antibody. Including doing.

In another aspect, the disclosure provides a method for inhibiting the activity of ApoC3 in a subject, the method comprising administering to the subject an effective amount of an antibody or pharmaceutical composition disclosed herein. including. In another aspect, the disclosure provides a method for reducing triglyceride levels in a subject's blood, which method administers an effective amount of the antibody or pharmaceutical composition disclosed herein to the subject. Including doing. In another aspect, the disclosure provides a method for inhibiting postprandial lipoemia in a subject, the method administering to the subject an effective amount of an antibody or pharmaceutical composition disclosed herein. Including that. In another aspect, the disclosure provides a method for treating hypertriglyceridemia in a subject, the method administering to the subject an effective amount of an antibody or pharmaceutical composition disclosed herein. Including that. In another aspect, the disclosure provides a method of treating chylomicronemia in a subject, the method comprising administering to the subject an effective amount of an antibody or pharmaceutical composition disclosed herein. include.

In another aspect, the disclosure provides a method for reducing the risk of cardiovascular disease in a subject suffering from hypertriglyceridemia, the method being disclosed to the subject in an effective amount herein. Includes administration of the antibody or pharmaceutical composition to be used. In certain embodiments, the cardiovascular disease is myocardial infarction. In certain embodiments, the cardiovascular disease is angina. In certain embodiments, the cardiovascular disease is stroke. In certain embodiments, the cardiovascular disease is atherosclerosis.

In certain embodiments of the aforementioned embodiments with respect to the method of treatment, the antibody reduces the level of chylomicrons or chylomicrons remnants in the blood of the subject. In certain embodiments, the subject is receiving an additional lipid lowering agent. In certain embodiments, the additional lipid lowering agent is an HMG-CoA reductase inhibitor. In certain embodiments, the HMG-CoA reductase inhibitor is atorvastatin, fluvastatin, lovastatin, pitavastatin, pravastatin, rosuvastatin or simvastatin. In certain embodiments, the additional lipid-lowering agent is a PCSK9 inhibitor. In certain embodiments, the PCSK9 inhibitor is alirocumab, evolocumab, or bocosizumab. In certain embodiments, the additional lipid-lowering agent is ezetimibe. In certain embodiments, the additional lipid-lowering agent is a combination of ezetimibe and an HMG-CoA reductase inhibitor. In certain embodiments, the additional lipid-lowering agent is a combination of ezetimibe, an HMG-CoA reductase inhibitor, and a PCSK9 inhibitor.

5E5WT (FIG. 1A), 5E5VH5_VL8 (FIG. 1B), and 5E5VHWT_VL8 (“VL8”), 5E5VH12_VLWT (“VH12”), 5E5VH5_VLWT (“VH5”), and 5E5VH5_VL8 (“VH5”), and 5E5VH5_VL8 (“VH5”), and 5E5VH5_VL8 (“VH5”) It is a series of graphs showing that the ability of HepG2 cells to inhibit the uptake of very low density lipoprotein (VLDL) was weakened. HepG2 cells were incubated with DiI VLDL and ApoC3 was purified either alone or in the presence of anti-ApoC3 antibody as shown. DiI VLDL ingested by HepG2 cells was measured by fluorescence spectroscopy of DiI dyes. HepG2 cells incubated with DiI VLDL alone (“VLDL”) acted as positive controls, and HepG2 cells incubated with DiI VLDL and purified ApoC3 were negative controls in the absence of anti-ApoC3 antibody (“ApoC3”). It worked as. 5E5WT (FIG. 1A), 5E5VH5_VL8 (FIG. 1B), and 5E5VHWT_VL8 (“VL8”), 5E5VH12_VLWT (“VH12”), 5E5VH5_VLWT (“VH5”), and 5E5VH5_VL8 (“VH5”), and 5E5VH5_VL8 (“VH5”), and 5E5VH5_VL8 (“VH5”) It is a series of graphs showing that the ability of HepG2 cells to inhibit the uptake of very low density lipoprotein (VLDL) was weakened. HepG2 cells were incubated with DiI VLDL and ApoC3 was purified either alone or in the presence of anti-ApoC3 antibody as shown. DiI VLDL ingested by HepG2 cells was measured by fluorescence spectroscopy of DiI dyes. HepG2 cells incubated with DiI VLDL alone (“VLDL”) acted as positive controls, and HepG2 cells incubated with DiI VLDL and purified ApoC3 were negative controls in the absence of anti-ApoC3 antibody (“ApoC3”). It worked as. 5E5WT (FIG. 1A), 5E5VH5_VL8 (FIG. 1B), and 5E5VHWT_VL8 (“VL8”), 5E5VH12_VLWT (“VH12”), 5E5VH5_VLWT (“VH5”), and 5E5VH5_VL8 (“VH5”), and 5E5VH5_VL8 (“VH5”), and 5E5VH5_VL8 (“VH5”) It is a series of graphs showing that the ability of HepG2 cells to inhibit the uptake of very low density lipoprotein (VLDL) was weakened. HepG2 cells were incubated with DiI VLDL and ApoC3 was purified either alone or in the presence of anti-ApoC3 antibody as shown. DiI VLDL ingested by HepG2 cells was measured by fluorescence spectroscopy of DiI dyes. HepG2 cells incubated with DiI VLDL alone (“VLDL”) acted as positive controls, and HepG2 cells incubated with DiI VLDL and purified ApoC3 were negative controls in the absence of anti-ApoC3 antibody (“ApoC3”). It worked as. FIG. 6 is a graph showing serum levels at various time points after injection of two anti-ApoC3 antibodies, 5E5 and 5E5VH5_VL8, and anti-chicken egg lysosomal human IgG ₁ antibody (HyHEL5) in an AAV8-human ApoC3 mouse model. It is a graph which shows the effect of 5E5 and 5E5VH5_VL8 on the level which circulates human ApoC3 in the AAV8-human ApoC3 mouse model. It is a graph which shows the effect of 5E5 and 5E5VH5_VL8 on the level which circulates ApoB in the AAV8-human ApoC3 mouse model. A series of surface plasmon resonance (SPR) sensorgrams showing the binding kinetics of 20 humanized anti-ApoC3 antibodies (mAb1-mAb20) to human ApoC3 when human ApoC3 is immobilized on a biosensor chip. Dotted and solid sensorgrams correspond to assay pH 7.4 and 5.5, respectively. For reference, the sensorgram corresponding to 5E5VH5_VL8 at a concentration of 25 nM is included in all graphs (dashed line at pH 7.4 and long dashed line at pH 5.5). A series of surface plasmon resonance (SPR) sensorgrams showing the binding kinetics of 20 humanized anti-ApoC3 antibodies (mAb1-mAb20) to human ApoC3 when human ApoC3 is immobilized on a biosensor chip. Dotted and solid sensorgrams correspond to assay pH 7.4 and 5.5, respectively. For reference, the sensorgram corresponding to 5E5VH5_VL8 at a concentration of 25 nM is included in all graphs (dashed line at pH 7.4 and long dashed line at pH 5.5). A series of surface plasmon resonance (SPR) sensorgrams showing the binding kinetics of 20 humanized anti-ApoC3 antibodies (mAb1-mAb20) to human ApoC3 when human ApoC3 is immobilized on a biosensor chip. Dotted and solid sensorgrams correspond to assay pH 7.4 and 5.5, respectively. For reference, the sensorgram corresponding to 5E5VH5_VL8 at a concentration of 25 nM is included in all graphs (dashed line at pH 7.4 and long dashed line at pH 5.5). A series of surface plasmon resonance (SPR) sensorgrams showing the binding kinetics of 20 humanized anti-ApoC3 antibodies (mAb1-mAb20) to human ApoC3 when human ApoC3 is immobilized on a biosensor chip. Dotted and solid sensorgrams correspond to assay pH 7.4 and 5.5, respectively. For reference, the sensorgram corresponding to 5E5VH5_VL8 at a concentration of 25 nM is included in all graphs (dashed line at pH 7.4 and long dashed line at pH 5.5). A series of surface plasmon resonance (SPR) sensorgrams showing the binding kinetics of 20 humanized anti-ApoC3 antibodies (mAb1-mAb20) to human ApoC3 when human ApoC3 is immobilized on a biosensor chip. Dotted and solid sensorgrams correspond to assay pH 7.4 and 5.5, respectively. For reference, the sensorgram corresponding to 5E5VH5_VL8 at a concentration of 25 nM is included in all graphs (dashed line at pH 7.4 and long dashed line at pH 5.5). A series of SPR sensorgrams showing the binding kinetics of 20 humanized anti-ApoC3 antibodies (mAb1 to mAb20) to human ApoC3 when the ApoC3 antibody is immobilized on a biosensor chip. Dotted and solid sensorgrams correspond to assay pH 7.4 and 5.5, respectively. For reference, the sensorgram corresponding to 5E5VH5_VL8 at a target nhuApoC3 concentration of 600 nM is included in all graphs (dashed pH 7.4 and long dashed line pH 5.5). A series of SPR sensorgrams showing the binding kinetics of 20 humanized anti-ApoC3 antibodies (mAb1 to mAb20) to human ApoC3 when the ApoC3 antibody is immobilized on a biosensor chip. Dotted and solid sensorgrams correspond to assay pH 7.4 and 5.5, respectively. For reference, the sensorgram corresponding to 5E5VH5_VL8 at a target nhuApoC3 concentration of 600 nM is included in all graphs (dashed pH 7.4 and long dashed line pH 5.5). A series of SPR sensorgrams showing the binding kinetics of 20 humanized anti-ApoC3 antibodies (mAb1 to mAb20) to human ApoC3 when the ApoC3 antibody is immobilized on a biosensor chip. Dotted and solid sensorgrams correspond to assay pH 7.4 and 5.5, respectively. For reference, the sensorgram corresponding to 5E5VH5_VL8 at a target nhuApoC3 concentration of 600 nM is included in all graphs (dashed pH 7.4 and long dashed line pH 5.5). A series of SPR sensorgrams showing the binding kinetics of 20 humanized anti-ApoC3 antibodies (mAb1 to mAb20) to human ApoC3 when the ApoC3 antibody is immobilized on a biosensor chip. Dotted and solid sensorgrams correspond to assay pH 7.4 and 5.5, respectively. For reference, the sensorgram corresponding to 5E5VH5_VL8 at a target nhuApoC3 concentration of 600 nM is included in all graphs (dashed pH 7.4 and long dashed line pH 5.5). A series of SPR sensorgrams showing the binding kinetics of 20 humanized anti-ApoC3 antibodies (mAb1 to mAb20) to human ApoC3 when the ApoC3 antibody is immobilized on a biosensor chip. Dotted and solid sensorgrams correspond to assay pH 7.4 and 5.5, respectively. For reference, the sensorgram corresponding to 5E5VH5_VL8 at a target nhuApoC3 concentration of 600 nM is included in all graphs (dashed pH 7.4 and long dashed line pH 5.5). FIG. 6 is a graph showing the percentage of activity of the selected humanized antibody and 5E5VH5_VL8 after incubating the antibody for 1 hour at the indicated temperature compared to the activity of the corresponding antibody incubated at 4 ° C. for 1 hour. The activity was measured as the association rate in FIGS. 5A and as the maximum RU (R0) in FIGS. 5B and 5C. FIG. 6 is a graph showing the percentage of activity of the selected humanized antibody and 5E5VH5_VL8 after incubating the antibody for 1 hour at the indicated temperature compared to the activity of the corresponding antibody incubated at 4 ° C. for 1 hour. The activity was measured as the association rate in FIGS. 5A and as the maximum RU (R0) in FIGS. 5B and 5C. FIG. 6 is a graph showing the percentage of activity of the selected humanized antibody and 5E5VH5_VL8 after incubating the antibody for 1 hour at the indicated temperature compared to the activity of the corresponding antibody incubated at 4 ° C. for 1 hour. The activity was measured as the association rate in FIGS. 5A and as the maximum RU (R0) in FIGS. 5B and 5C.

The present disclosure provides antibodies that specifically bind to ApoC3 (eg, human ApoC3) and inhibit ApoC3 function. Pharmaceutical compositions containing these antibodies, nucleic acids encoding these antibodies, expression vectors and host cells for making these antibodies, and methods of treating a subject with these antibodies are also provided. In certain embodiments, the anti-ApoC3 antibodies disclosed herein can diminish the ability of ApoC3 to inhibit the uptake of TRL by hepatocytes, and when administered to a subject, the serum levels of ApoC3 and ApoB. It can be reduced rapidly and persistently. Therefore, the disclosed anti-ApoC3 antibodies are useful in the treatment and prevention of hypertriglyceridemia and related diseases (eg, cardiovascular disease and pancreatitis).

1. 1. Definitions As used herein, the term "ApoC3" refers to the apolipoprotein C3 protein. In certain embodiments, the ApoC3 is a human ApoC3. An exemplary human ApoC3 amino acid sequence is shown in RefSeq Accession No. NP_000031.1. The mature amino acid sequence of NP_000031.1 is as follows.
SEAEDASLLSFMQGYMKHATKTAKDALSSVQESQVAQQARGGWVTDGFSSLKDYWSTVKDKFSEFWDLDPEVRPTSAVA (SEQ ID NO: 1).

As used herein, the terms "antibody" and "multiple antibodies" include full-length antibodies, antigen-binding fragments of full-length antibodies, and molecules containing the CDR, VH or VL regions of an antibody. Examples of antibodies include monoclonal antibodies, recombinant antibodies, monospecific antibodies, multispecific antibodies (including bispecific antibodies), human antibodies, humanized antibodies, chimeric antibodies, immunoglobulins, synthetic antibodies, etc. A tetramer antibody containing two heavy chains and two light chain molecules, an antibody light chain monomer, an antibody heavy chain monomer, an antibody light chain dimer, an antibody heavy chain dimer, an antibody light chain-anti-antibody. Weight chain pair, intrabody, heteroconjugate antibody, single domain antibody, monovalent antibody, single chain antibody or single chain Fv (scFv), scFv-Fc, camel antibody (eg, llama antibody), camelized antibody, affi Includes body, Fab fragment, F (ab') ₂ fragment, disulfide-bound Fv (sdFv), anti-idiotype (anti-Id) antibody (including, for example, anti-anti-Id antibody), and any of the above antigen-binding fragments. Is done. In certain embodiments, the antibodies disclosed herein refer to a polyclonal antibody population. Antibodies can be any type of immunoglobulin molecule (eg, IgG, IgE, IgM, IgD, IgA, or IgY), any class (eg, IgG ₁ , IgG ₂ , IgG ₃ , IgG ₄ , IgA ₁ , or IgA). ₂ ), or any subclass (eg, IgG _2a or IgG _2b ). In certain embodiments, the antibodies disclosed herein are IgG antibodies, or a class thereof (eg, human IgG ₁ or IgG ₄ ) or a subclass thereof. In one particular embodiment, the antibody is a humanized monoclonal antibody.

As used herein, the term "isolated antibody" refers to an antibody that has been identified and isolated and / or recovered from at least one component of its natural environment. The term "isolated antibody" includes an in situ antibody in a recombinant host cell.

As used herein, the terms "CDR" or "complementarity determining regions" mean non-adjacent antigen binding sites found within the variable regions of both heavy and light chain polypeptides. These specific regions are described in Kabat et al. , J. Biol. Chem. 252, 6609-6616 (1977) and Kabat et al. , Sequences of protein of immunological intervention. According to (1991), Chothia et al. , J. Mol. Biol. 196: by 901-917 (1987), as well as MacCallum et al. , J. Mol. Biol. 262: 732-745 (1996), all of which are incorporated by reference in their entirety, and when compared to each other, the definition includes overlapping or subset of amino acid residues. In certain embodiments, the term "CDR" is used in Kabat et al. , J. Biol. Chem. 252, 6609-6616 (1977) and Kabat et al. , Sequences of protein of immunological intervention. CDR, as defined by (1991). CDRH1, CDRH2, and CDRH3 represent heavy chain CDRs, and CDRL1, CDRL2, and CDRL3 represent light chain CDRs.

As used herein, the term "framework (FR) amino acid residue" refers to an amino acid in the framework region of an immunoglobulin chain. As used herein, the term "framework region" or "FR region" is part of a variable region, but not part of a CDR (eg, using the Kabat definition of a CDR). Contains amino acid residues.

As used herein, the terms "variable domain" and "variable domain" are used interchangeably and are common in the art. A variable region is typically a portion of an antibody, generally a light chain or a portion of a heavy chain, typically about 110-120 amino acids or 110-125 amino acids and a mature light chain at the amino end of the mature heavy chain. Refers to about 90-115 amino acids in, which are used in the binding and specificity of a particular antibody to those particular antigens, where the sequences differ widely among the antibodies. The variability in the sequence is concentrated in such regions called complementarity determining regions (CDRs), while the more highly conserved regions in the variable domain are called framework regions (FRs). Although not bound by any particular mechanism or theory, light chain and heavy chain CDRs are primarily involved in the interaction and specificity of the antibody with the antigen. In certain embodiments, the variable region is a human variable region. In certain embodiments, the variable region comprises a rodent or mouse CDR and a human framework region (FR). In certain embodiments, the variable region is a primate (eg, non-human primate) variable region. In certain embodiments, the variable region comprises a rodent or mouse CDR and a primate (eg, non-human primate) framework region (FR).

The terms "VL" and "VL domain" are used interchangeably to refer to the light chain variable region of an antibody.

The terms "VH" and "VH domain" are used interchangeably to refer to the heavy chain variable region of an antibody.

As used herein, the terms "stationary domain" and "constant domain" are interchangeable and common in the art. The constant region is the antibody moiety, eg, the carboxyl-terminal portion of the light or heavy chain that is not directly involved in the binding of the antibody to the antigen but can exhibit various effector functions such as interaction with Fc receptors. be. The constant region of an immunoglobulin molecule generally has more conserved amino acid sequences than the immunoglobulin variable domain.

As used herein, the term "heavy chain", when used with reference to an antibody, is based on the amino acid sequence of a constant domain and can be of any different type, eg, alpha (α), delta (δ). ), Epsilon (ε), gamma (γ), and muμ), from which the antibodies IgA, IgD, IgE, IgG, and IgM classes (IgG subclasses such as IgG ₁ , IgG ₂ , IgG ₃ ). , And IgG ₄ ), respectively.

As used herein, the term "light chain", when used with reference to an antibody, is based on the amino acid sequence of the constant domain and is of any different type, eg kappa (κ) or lambda (λ). ) Can be pointed out. Light chain amino acid sequences are well known in the art. In certain embodiments, the light chain is a human light chain.

As used herein, the term "EU position" is used by Edelman, G. et al. M. et al, Proc. Natl. Acad. USA, 63, 78-85 (1969), and Kabat et al, in "Sequences of Proteins of Immunological Interest", U.S.A. S. Dept. As described in Health and Human Services, 5th edition, 1991, refers to amino acid positions according to EU numbering conventions for constant regions of an antibody, each of which is incorporated herein by reference in its entirety.

As used herein, the term "specifically binds to" is less than about 1 x ^10-6 M, 1 x ^10-7 M, 1 x ^10-8 M, 1 x ^10-9 M. It binds to an antigen with a dissociation constant (KD) of 1, 1 × 10 ^-10 _M , 1 × 10 ^-11 M, 1 × 10 ^-12 M, or less, or more than its affinity for non-specific antigens. Refers to the ability of an antibody to bind an antigen with an affinity that is at least twice as great.

As used herein, "epitope" refers to a localized region of an antigen to which an antibody can specifically bind. The epitope can be, for example, an adjacent amino acid (linear or adjacent epitope) of the polypeptide, or the epitope can be, for example, two or more non-adjacent regions (three-dimensional structure, non-linear, discontinuous,) of the polypeptide (s). Or it can be formed from non-adjacent epitopes). In certain embodiments, the epitope to which the antibody binds is, for example, NMR spectroscopy, X-ray diffraction crystallography studies, ELISA assay, hydrogen / deuterium exchange with mass spectrometry (eg, liquid chromatography electrospray mass spectrometry). It can be determined by peptide scanning assay or mutagenesis mapping (eg, site-specific mutagenesis mapping).

As used herein, the terms "treat," "treat," and "treat" refer to the therapeutic or prophylactic measures disclosed herein. The method of "treatment" is the risk of preventing, curing, delaying, reducing the severity of, or developing one or more symptoms of the disease or disorder, or recurrent disease or disorder. To alleviate or improve, or to prolong the survival of a subject expected in the absence of such treatment, to resist a subject who has or is susceptible to the disease or disorder. Administration of ApoC3 antibody is used.

As used herein, the term "effective amount" refers to the amount of therapeutic agent that achieves the desired prophylactic or therapeutic effect in the context of administration of the therapeutic agent to a subject.

As used herein, the term "subject" includes any human or non-human animal.

As used herein, the term "or" means and / or.

As used herein, the terms "about" and "approximately", when used to modify a numerical or numerical range, exceed the value or range by 5% to 10%, and 5% to. A deviation of less than 10% indicates that the quoted value or range is within the intended meaning of the range.

2. 2. Anti-ApoC3 Antibodies The present disclosure provides isolated antibodies (eg, humanized antibodies) that specifically bind to ApoC3 (eg, human ApoC3) and inhibit ApoC3 function.

In certain embodiments, the isolated antibody binds to the mammalian ApoC3 protein. In certain embodiments, the isolated antibody binds to human ApoC3. In certain embodiments, the isolated antibody binds to Macafascillaris ApoC3.

In certain embodiments, the isolated antibody has a higher affinity for ApoC3 (eg, human ApoC3) at a physiological pH (eg, pH 7.4) than under acidic pH (eg, pH 5.5-pH 6). Combine by sex. Methods for producing such pH-dependent antibodies are well known in the art. For example, in one exemplary method, one or more amino residues in the heavy and / or light chain CDRs of an anti-ApoC3 antibody can be found in Igawa et al. , Nat Biotechnology. (2010) 28 (11), 1203-1207, Chaparro-Riggers et al. , J BiolChem. (2012) 287 (14): Replaced with histidine residues as described in US Pat. No. 9,090,651, and US Pat. No., 2011/0111406A1, each of which is replaced with a histidine residue. The whole is incorporated herein by reference. However, such methods are well known in the art, but those skilled in the art will appreciate any given antibody in order to achieve pH-dependent binding to the antigen without disrupting the antibody's affinity for the antigen. Understand that the exact CDR amino acid that can be mutated to histidine can only be determined empirically (see, eg, Edgcomb and Murphy, Proteins (2002) 49: 1-6, which can be determined by reference. The whole is incorporated herein).

Those skilled in the art can understand that the affinity of an antibody for an antigen can be indicated by the dissociation constant ( _KD ), and the smaller the _KD , the higher the affinity. Thus, in certain embodiments, the anti- _ApoC3 antibody binds to _ApoC3 (eg, human _ApoC3 ) at a first KD at pH 7.4 and a second KD at pH 5.5 and with a second KD. The ratio of the first _KD is at least 1 (eg, at least 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9). 2,2.5,3,3.5,4,4.5,5,6,7,8,9,10,11,12,13,14,15,20,25,30,35,40 , 45, 50, 60, 70, 80, 90, or 100).

In certain embodiments, the first _KD is less than 100 nM (eg, less than 50, 20, 10, 5, 2, 1, 0.5, 0.2, or 0.1 nM). In certain embodiments, the second _KD is greater than 1 nM (eg, greater than 2, 5, 10, 20, or 50 nM, or 0.1, 0.2, 0.5, 1, 2, 5). Over 10, 20, 50, or 100 μM). In certain embodiments, the first KD is less than 100 nM (eg, less than 50, 20, 10, 5, 2, 1, 0.5, 0.2, or 0.1 nM), and _ApoC3 (eg, less than 0.1 nM). Antibodies in animals expressing human ApoC3) (eg, humans or mice) have a half-life of at least about 1 day (eg, at least about 2, 3, 4, 5, 6, or 7 days, or about 1, More than 2, 3, 4, 6, or 8 weeks). In certain embodiments, the ApoC3 is a human ApoC3 and the animal expressing ApoC3 is a human. In certain embodiments, ApoC3 is human ApoC3 and the animal expressing ApoC3 is a mouse expressing human ApoC3.

In certain embodiments, the isolated antibodies disclosed herein reduce the ability of ApoC3 to inhibit hepatocyte uptake of TRL (eg, VLDL) or TRL remnants (in vivo or in vitro). In certain embodiments, the isolated antibodies disclosed herein are TRL (eg, VLDL) or TRL when evaluated by the methods disclosed herein or by methods known to those of skill in the art. ApoC3's ability to inhibit remnant hepatocyte uptake is at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%. , 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% weakening. In certain embodiments, the isolated antibodies disclosed herein are TRL (eg, VLDL) or TRL when evaluated by the methods disclosed herein or by methods known to those of skill in the art. The ability of ApoC3 to inhibit the uptake of remnant hepatocytes is at least about 1.1 times, 1.2 times, 1.3 times, 1.4 times, 1.5 times, 2 times, 2.5 times, 3 times. Double, 3.5 times, 4 times, 4.5 times, 5 times, 6 times, 7 times, 8 times, 9 times, 10 times, 15 times, 20 times, 30 times, 40 times, 50 times, 60 times , 70 times, 80 times, 90 times, or 100 times weaker.

In certain embodiments, the isolated antibodies disclosed herein can inhibit postprandial lipoemia in a subject when administered to the subject before, during, or after a meal. In certain embodiments, the anti-ApoC3 antibodies disclosed herein have at least 5% postprandial lipoemia in a subject when evaluated by the methods disclosed herein or by methods known to those of skill in the art. 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90 %, 95%, 98%, or 99% can be inhibited. In certain embodiments, the anti-ApoC3 antibodies disclosed herein will cause at least about 1 postprandial lipoemia in a subject when evaluated by the methods disclosed herein or by methods known to those of skill in the art. .1x, 1.2x, 1.3x, 1.4x, 1.5x, 2x, 2.5x, 3x, 3.5x, 4x, 4.5x, 5x , 6 times, 7 times, 8 times, 9 times, 10 times, 15 times, 20 times, 30 times, 40 times, 50 times, 60 times, 70 times, 80 times, 90 times, or 100 times. can.

In certain embodiments, the isolated antibodies disclosed herein reduce postprandial chylomicron levels or chylomicron remnant levels in a subject when administered to the subject before, during, or after a meal. Can be done. In certain embodiments, the anti-ApoC3 antibodies disclosed herein are postprandial chylomicron levels or chylomicrons remnants in a subject when evaluated by the methods disclosed herein or by methods known to those of skill in the art. Level at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80% , 85%, 90%, 95%, 98%, or 99%. In certain embodiments, the anti-ApoC3 antibodies disclosed herein are postprandial chylomicron levels or chylomicrons remnants in a subject when evaluated by the methods disclosed herein or by methods known to those of skill in the art. Levels are at least about 1.1 times, 1.2 times, 1.3 times, 1.4 times, 1.5 times, 2 times, 2.5 times, 3 times, 3.5 times, 4 times, 4. 5 times, 5 times, 6 times, 7 times, 8 times, 9 times, 10 times, 15 times, 20 times, 30 times, 40 times, 50 times, 60 times, 70 times, 80 times, 90 times, or 100 times. It can be doubled.

In certain embodiments, the isolated antibodies disclosed herein can increase the rate of clearance of ApoC3 and / or ApoB (eg, ApoB48 and / or ApoB100) from the blood of interest. In certain embodiments, the anti-ApoC3 antibody is ApoC3 and / or ApoB (eg, ApoB48 and / or) from the blood of interest when evaluated by the methods disclosed herein or by methods known to those of skill in the art. ApoB100) clearance rates of at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, It can be increased by 75%, 80%, 85%, 90%, 95%, 98%, or 99%. In certain embodiments, the anti-ApoC3 antibodies disclosed herein are ApoC3 and / or ApoB from the blood of interest when evaluated by the methods disclosed herein or by methods known to those of skill in the art. Clearance rates of (eg, ApoB48 and / or ApoB100) are at least about 1.1x, 1.2x, 1.3x, 1.4x, 1.5x, 2x, 2.5x, 3 Double, 3.5 times, 4 times, 4.5 times, 5 times, 6 times, 7 times, 8 times, 9 times, 10 times, 15 times, 20 times, 30 times, 40 times, 50 times, 60 times , 70 times, 80 times, 90 times, or 100 times. Methods for assessing clearance of ApoC3 and / or ApoB (eg, ApoB48 and / or ApoB100) include, but are not limited to, isotope tracer techniques, where isotopes are either radioactive or stable. May be.

In certain embodiments, the isolated antibodies disclosed herein can reduce the levels of ApoC3 and / or ApoB (eg, ApoB48 and / or ApoB100) in the blood of a subject. In certain embodiments, the anti-ApoC3 antibody is ApoC3 and / or ApoB (eg, ApoB48 and / or) in the blood of subject when evaluated by the methods disclosed herein or by methods known to those of skill in the art. ApoB100) levels of at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%. , 80%, 85%, 90%, 95%, 98%, or 99%. In certain embodiments, the anti-ApoC3 antibodies disclosed herein are ApoC3 and / or in the blood of subject when evaluated by the methods disclosed herein or by methods known to those of skill in the art. Levels of ApoB (eg, ApoB48 and / or ApoB100) are at least about 1.1x, 1.2x, 1.3x, 1.4x, 1.5x, 2x, 2.5x, 3x. , 3.5 times, 4 times, 4.5 times, 5 times, 6 times, 7 times, 8 times, 9 times, 10 times, 15 times, 20 times, 30 times, 40 times, 50 times, 60 times, It can be reduced by 70 times, 80 times, 90 times, or 100 times. In certain embodiments, the reduction in levels of ApoC3 and / or ApoB (eg, ApoB48 and / or ApoB100) in the blood of a subject is at least 1, 2, 3, 4, 5, 6, 7, 8, 9, ,. It is maintained for 10, 15, 20, 25, 30, 35, 40, 45, or 50 days, or at least 1, 2, 3, 4, 5, 6, 7, or 8 weeks.

In certain embodiments, the isolated antibody disclosed herein can bind to a lipid-bound ApoC3 (eg, triglyceride, TRL (eg, VLDL), or ApoC3 bound to a TRL remnant). In certain embodiments, the isolated antibodies disclosed herein do not inhibit the binding of ApoC3 to lipids or lipoproteins. In certain embodiments, the antibodies disclosed herein do not compete for binding of ApoC3 to lipids or lipoproteins. In certain embodiments, the lipid comprises a fatty acid chain. In certain embodiments, the lipid comprises a phosphatidyl group. In certain embodiments, the lipid comprises phosphatidylcholine (eg, DMPC), phosphatidylserine, phosphatidylethanolamine, phosphatidylinositol, or phosphatidylglycerol. In certain embodiments, the lipid is a triglyceride. In certain embodiments, the lipoprotein is TRL (eg, VLDL) or TRL remnant. In certain embodiments, the ability of ApoC3 to bind lipids and lipoproteins (eg, triglycerides, TRL (eg, VLDL) or TRL remnants) in the presence of the anti-ApoC3 antibodies disclosed herein is described herein. At least 50%, 55%, 60% of the ability of ApoC3 to bind to the same lipids and lipoproteins in the absence of anti-ApoC3 antibodies, as assessed by the methods disclosed in the above, or by methods known to those of skill in the art. %, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99%.

In certain embodiments, the isolated antibodies disclosed herein reduce the ability of ApoC3 to inhibit hepatocyte uptake of TRL (eg, VLDL) or TRL remnants. In certain embodiments, uptake of TRL (eg, VLDL) or TRL remnant by hepatocytes (eg, HepG2 cells) in the presence of anti-ApoC3 antibodies disclosed herein is in the absence of anti-ApoC3 antibodies. At least 1.1, 1.2, 1.3, 1.4, 1.5, 1.6 than uptake of TRL (eg, VLDL) or TRL remnant by hepatocytes (eg, HepG2 cells). 1.7, 1.8, 1.9, 2, 2.5, 3, 3.5, 4, 4.5, or 5 times higher.

In certain embodiments, the isolated antibodies disclosed herein weaken the ability of ApoC3 to inhibit hepatocyte uptake of TRL (eg, VLDL) or TRL remnants, and lipid-bound ApoC3 (eg, eg). It can bind to triglycerides, TRL (eg, VLDL), or ApoC3 bound to TRL remnants.

In certain embodiments, the isolated antibody disclosed herein binds to an epitope of ApoC3 within the amino acid sequence FSEFWDLDPE (SEQ ID NO: 2). In certain embodiments, the epitope comprises at least one amino acid within SEQ ID NO: 2, optionally including one or more amino acids from SEQ ID NO: 1 flanking SEQ ID NO: 2. In certain embodiments, the epitope comprises at least one amino acid at position 2, 5, 6, 8, or 10 of SEQ ID NO: 2. In certain embodiments, the epitope comprises at least two amino acids at positions 2, 5, 6, 8, or 10 of SEQ ID NO: 2. In certain embodiments, the epitope comprises at least three amino acids at positions 2, 5, 6, 8, or 10 of SEQ ID NO: 2. In certain embodiments, the epitope comprises at least 4 amino acids at positions 2, 5, 6, 8, or 10 of SEQ ID NO: 2. In certain embodiments, the epitope comprises an amino acid at positions 5 and 6 of SEQ ID NO: 2. In certain embodiments, the epitope comprises the amino acids at positions 2, 5, and 6 of SEQ ID NO: 2. In certain embodiments, the epitope comprises an amino acid at positions 2, 5, and 8 of SEQ ID NO: 2. In certain embodiments, the epitope comprises an amino acid at positions 2, 5, 6, and 8 of SEQ ID NO: 2. In certain embodiments, the epitope comprises an amino acid at position 10 of SEQ ID NO: 2. In certain embodiments, the epitope comprises an amino acid at positions 6 and 10 of SEQ ID NO: 2. In certain embodiments, the epitope comprises an amino acid at positions 8 and 10 of SEQ ID NO: 2. In certain embodiments, the epitope comprises an amino acid at positions 6 and 8 of SEQ ID NO: 2. In certain embodiments, the epitope comprises an amino acid at positions 6, 8, and 10 of SEQ ID NO: 2. In certain embodiments, the antibody can bind to lipid-bound ApoC3 (eg, triglycerides, TRL (eg, VLDL), or ApoC3 bound to TRL remnants). In certain embodiments, the antibody cannot diminish the ability of ApoC3 to inhibit lipoprotein lipase-mediated lipid degradation of TRL (eg, VLDL). In certain embodiments, the antibody also diminishes the ability of ApoC3 to inhibit hepatocyte uptake of TRL (eg, VLDL) or TRL remnant. In certain embodiments, the antibody can also inhibit postprandial lipoemia in a subject when administered to the subject before, during, or after a meal. In certain embodiments, the antibodies disclosed herein can also reduce postprandial chylomicron levels or chylomicron remnant levels in a subject when administered to the subject before, during, or after a meal.

Any suitable assay can be used to measure the aforementioned functional activity of the antibodies disclosed herein. Exemplary assays include, but are not limited to, the functional assays disclosed in the examples herein.

The amino acid sequences of exemplary anti-ApoC3 antibodies are shown in Tables 1-13 herein.

The amino acid sequences of exemplary humanized anti-ApoC3 antibodies are shown in Tables 8-13 herein.

In certain embodiments, the present disclosure provides an isolated antibody that specifically binds to ApoC3 (eg, human ApoC3), which antibody is the VH domain set forth in Table 3 or 10 herein. Includes a VH domain that includes one, two, or all three of the CDRs. In certain embodiments, the antibody comprises CDRH1 of one of the VH domains shown in Table 3 or 10. In certain embodiments, the antibody comprises CDRH2, one of the VH domains shown in Table 3 or 10. In certain embodiments, the antibody comprises CDRH3, one of the VH domains shown in Table 3 or 10.

In certain embodiments, the present disclosure provides an isolated antibody that specifically binds to ApoC3 (eg, human ApoC3), which antibody is the VL domain disclosed in Table 4 or 11 herein. Includes a VL domain containing one, two, or all three of the CDRs of. In certain embodiments, the antibody comprises CDRL1 of one of the VL domains shown in Table 4 or 11. In certain embodiments, the antibody comprises CDRL2, one of the VL domains shown in Table 4 or 11. In certain embodiments, the antibody comprises CDRL3, one of the VL domains shown in Table 4 or 11.

In certain embodiments, the present disclosure provides an isolated antibody that specifically binds to ApoC3 (eg, human ApoC3), the antibody being a heavy chain having complementarity determining regions CDRH1, CDRH2, and CDRH3. The antibodies shown in Table 5 or 12, which include variable regions and light chain variable regions having complementarity determining regions CDRL1, CDRL2, and CDRL3, are CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3, respectively. , CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3 amino acid sequences.

In certain embodiments, the CDRs of the antibody are described in Kabat et al. , J. Biol. Chem. 252, 6609-6616 (1977) and Kabat et al. , Sequences of protein of immunological intervention (1991). In certain embodiments, the light chain CDRs of the antibody are determined according to Kabat and the heavy chain CDRs of the antibody are determined according to MacCallum (above).

In certain embodiments, the CDR of an antibody can be determined according to a Chothia numbering scheme pointing to the location of an immunoglobulin structure loop (eg, Chothia Chothia C & Lesk AM, (1987), J Mol Biol 196: 901-917, Al-Lazikani B et al., (1997) J Mol Biol 273: 927-948, Chothia C et al, (1992) J Mol Biol 227: 799-817, Tramontano A et al., (1990) J Mol. (1): 175-82, and US Pat. No. 7,709,226). Typically, when using the Kabat numbering rule, the Chothia CDRH1 loop is present at heavy chain amino acids 26-32, 33, or 34, and the Chothia CDRH2 loop is present at heavy chain amino acids 52-56, Chothia. The CDRH3 loop is present at heavy chain amino acids 95-102, while the Chothia CDRL1 loop is present at light chain amino acids 24-34, the Chothia CDRL2 loop is present at light chain amino acids 50-56, and the Chothia CDRL3 loop is present at light chain amino acids 50-56. It is present in light chain amino acids 89-97. The ends of the Chothia CDRH1 loop when numbered using the Kabat numbering rule vary between H32 and H34 depending on the length of the loop (this is because the Kabat numbering scheme inserts into H35A and H35B). Because of the placement; if neither 35A nor 35B is present, the loop ends at 32; if only 35A is present, the loop ends at 33; if both 35A and 35B are present, the loop ends at 34. Ends with).

In certain embodiments, the CDRs of the antibodies are described in Lefranc MP, (1999) The Immunologist 7: 132-136 and Lefranc MP et al. , (1999) Nucleic Acids Res 27: 209-212, can be determined according to the IMGT numbering system. According to the IMGT numbering scheme, CDRH1 is in the 26th to 35th positions, CDRH2 is in the 51st to 57th positions, CDRH3 is in the 93rd to 102nd positions, CDRL1 is in the 27th to 32nd positions, and CDRL2 is in the 27th to 32nd positions. It is in the 50th to 52nd positions, and CDRL3 is in the 89th to 97th positions.

In certain embodiments, the CDRs of an antibody can be determined according to an AbM numbering scheme pointing to the AbM hypervariable region, which represents a compromise between the Kabat CDR and the Chothia structural loop, Oxford Modeler's. Used by AbM antibody modeling software (Oxford Molecular Group, Inc.).

In certain embodiments, the CDRs of the antibody are described in MacCalllum RM et al. , (1996) J Mol Biol 262: 732-745. For example, Antibody Engineering, Kontermann and Dubel, eds. , Chapter 31, pp. 422-439, Springer-Verlag, Berlin (2001), Martin A. et al. See also "Protein Sequence and Structure Analysis of Antibodies Variable Domains".

In certain embodiments, the present disclosure provides an isolated antibody that specifically binds to ApoC3 (eg, human ApoC3), wherein the antibodies are CDRH1, CDRH2, and CDRH3 of the VH domains shown in Table 3. A heavy chain variable region comprising a regional amino acid sequence and a light chain variable region comprising the CDRL1, CDRL2, and CDRL3 region amino acid sequences of the VL domains shown in Table 4 are included, each CDR independently herein. As disclosed, it is defined according to the definition of CDR by Kabat, Chothia, IMGT, MacCallum, or AbM.

In certain embodiments, the present disclosure provides isolated antibodies (eg, humanized antibodies) that specifically bind to ApoC3 (eg, human ApoC3), wherein the antibodies are complementarity determining regions CDRH1, CDRH2. , And a heavy chain variable region with CDRH3 and a light chain variable region with complementarity determining regions CDRL1, CDRL2, and CDRL3.
(A) CDRH1 comprises the amino acid sequence of TYSMR (SEQ ID NO: 3).
(B) CDRH 2 is SIX ₁ TX ₂ X ₃ GGTAYRDSVKG (in the formula, X ₁ is S or H, X ₂ is G, E, or D, and X ₃ is G or A. ) (SEQ ID NO: 93) containing the amino acid sequence
(C) CDRH 3 comprises the amino acid sequence of X ₄ GYSD (where X ₄ is A or H in the formula) (SEQ ID NO: 5).
(D) CDRL1 comprises the amino acid sequence of KTSQGLVHSX ₅ GKTYFY (where _X5 is D or G in the formula) (SEQ ID NO: 88).
(E) CDRL2 comprises the amino acid sequence of QVSNRAS (SEQ ID NO: 7), and (f) CDRL3 is the amino acid sequence of AX ₆ GTYYPHT (where X ₆ is Q or H in the formula) (SEQ ID NO: 8). Including
In the formula, at least one of X ₁ , X ₄ , and X ₆ is H.

In certain embodiments, the CDRH2 is SIHTGGGTAYRDSVKG (SEQ ID NO: 36), SIHTGAGTAYRDSVKG (SEQ ID NO: 37), SIHTDAGGTAYRDSVKG (SEQ ID NO: 38), SIHTGGGTAYRDSVKG (SEQ ID NO: 39), SISTDGGTAYRDSVKG (SEQ ID NO: 39), SISTDGGTAY. ) Contains the amino acid sequence.

In certain embodiments, CDRH3 comprises the amino acid sequence of AGYSD (SEQ ID NO: 10) or HGYSD (SEQ ID NO: 12).

In certain embodiments, CDRL1 comprises the amino acid sequence of KTSQGLVHSDGKTYFY (SEQ ID NO: 6) or KTSQGLVHSGGKTYFY (SEQ ID NO: 40).

In certain embodiments, CDRL3 comprises the amino acid sequence of AHGTYYPHT (SEQ ID NO: 14) or AQGTYYPHT (SEQ ID NO: 13).

In certain embodiments, the present disclosure provides an isolated antibody (eg, a humanized antibody) that specifically binds to ApoC3 (eg, human ApoC3), wherein the antibodies are SEQ ID NOs: 3, 36, respectively. , And 10; 3,37, and 10; 3,38, and 10; 3,39, and 10; 3,9, and 10; 3,11, and 10; 3,9,12; or 3,11, And 12 comprises a VH domain comprising the amino acid sequences of CDRH1, CDRH2, and CDRH3 shown in 12.

In certain embodiments, the present disclosure provides an isolated antibody that specifically binds to ApoC3 (eg, human ApoC3), wherein the antibodies are SEQ ID NOs: 6, 7, and 14; 40, 7, respectively. , And 14; 40, 7, and 13; or VL domains comprising the amino acid sequences of CDRL1, CDRL2, and CDRL3 shown in 6, 7, and 13.

In certain embodiments, the present disclosure is an isolated antibody (eg, a humanized antibody) that specifically binds to ApoC3 (eg, human ApoC3), with SEQ ID NOs: 42, 43, 44, 45, 46. , 47, 48, 49, 50, 51, 52, 53, 15, 16, 17, or 18 and at least 75%, 80%, 85%, 90%, 95%, or 100% (eg,). , At least 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99%) isolated, comprising heavy chain variable regions containing identical amino acid sequences. Provide antibodies. In certain embodiments, the antibody has the amino acid sequence set forth in SEQ ID NO: 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 15, 16, 17, or 18. Includes heavy chain variable region.

In certain embodiments, the present disclosure is an isolated antibody (eg, a humanized antibody) that specifically binds to ApoC3 (eg, human ApoC3), with SEQ ID NOs: 54, 55, 56, 57, 58. , 59, 60, 61, 62, 63, 64, 65, 19, or 20 and at least 75%, 80%, 85%, 90%, 95%, or 100% (eg, at least 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99%) provides an isolated antibody comprising a heavy chain variable region comprising an amino acid sequence that is identical. .. In certain embodiments, the antibody is a light chain variable region having the amino acid sequence set forth in SEQ ID NO: 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 19, or 20. including.

In certain embodiments, the present disclosure is an isolated antibody (eg, a humanized antibody) that specifically binds to ApoC3 (eg, human ApoC3), with SEQ ID NOs: 42, 43, 44, 45, 46. , 47, 48, 49, 50, 51, 52, 53, 15, 16, 17, or 18 and at least 75%, 80%, 85%, 90%, 95%, or 100% (eg,). , At least 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99%) Heavy chain variable regions containing identical amino acid sequences and SEQ ID NOs: 54, 55. , 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 19, or 20 and at least 75%, 80%, 85%, 90%, 95%, or 100%. (Eg, at least 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99%) comprising a light chain variable region comprising an identical amino acid sequence. An isolated antibody is provided. In certain embodiments, the antibody has the amino acid sequence set forth in SEQ ID NO: 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 15, 16, 17, or 18. A heavy chain variable region and a light chain variable region having the amino acid sequence set forth in SEQ ID NO: 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 19, or 20. include.

In certain embodiments, the present disclosure provides isolated antibodies (eg, humanized antibodies) that specifically bind to ApoC3 (eg, human ApoC3), wherein the antibodies are complementarity determining regions CDRH1, CDRH2. , And a heavy chain variable region with CDRH3 and a light chain variable region with complementarity determining regions CDRL1, CDRL2, and CDRL3.
(A) CDRH1 comprises the amino acid sequence of TYSMR (SEQ ID NO: 3).
(B) CDRH 2 comprises the amino acid sequence of SIHTX ₁ X ₂ GGTAYRDSVKG (where X ₁ is G, E, or D and X ₂ is G or A) (SEQ ID NO: 87).
(C) CDRH3 comprises the amino acid sequence of AGYSD (SEQ ID NO: 10).
(D) CDRL1 comprises the amino acid sequence of KTSQGLVHSXGKTYFY (where X is D or G in the formula) (SEQ ID NO: 88).
(E) CDRL2 comprises the amino acid sequence of QVSNRAS (SEQ ID NO: 7), and (f) CDRL3 comprises the amino acid sequence of AXGTYYPHT (where X is Q or H in the formula) (SEQ ID NO: 8).

In certain embodiments, the present disclosure provides isolated antibodies (eg, humanized antibodies) that specifically bind to ApoC3 (eg, human ApoC3), wherein the antibodies are complementarity determining regions CDRH1, CDRH2. , And a heavy chain variable region with CDRH3 and a light chain variable region with complementarity determining regions CDRL1, CDRL2, and CDRL3.
(A) CDRH1 comprises the amino acid sequence of TYSMR (SEQ ID NO: 3).
(B) CDRH2 comprises the amino acid sequence of SIHTGGGTAYRDSVKG (SEQ ID NO: 36), SIHTAGEGGTAYRDSVKG (SEQ ID NO: 37), SIHTDAGGTAYRDSVKG (SEQ ID NO: 38), or SIHTEGGTAYRDSVKG (SEQ ID NO: 39).
(C) CDRH3 comprises the amino acid sequence of AGYSD (SEQ ID NO: 10).
(D) CDRL1 comprises the amino acid sequence of KTSQGLVHSDGKTYFY (SEQ ID NO: 6) or KTSQGLVHSGGKTYFY (SEQ ID NO: 40).
(E) CDRL2 comprises the amino acid sequence of QVSNRAS (SEQ ID NO: 7), and (f) CDRL3 comprises the amino acid sequence of AHGTYYPHT (SEQ ID NO: 14) or AQGTYYPHT (SEQ ID NO: 13).

In certain embodiments, CDRH2 comprises the amino acid sequence of SIHTGGGTAYRDSVKG (SEQ ID NO: 36), SIHTAGEGGTAYRDSVKG (SEQ ID NO: 37), SIHTDAGGTAYRDSVKG (SEQ ID NO: 38), or SIHTGGGTAYRDSVKG (SEQ ID NO: 39). In certain embodiments, CDRH2 comprises the amino acid sequence of SIHTGGGTAYRDSVKG (SEQ ID NO: 36), SIHTEAGGTAYRDSVKG (SEQ ID NO: 37), or SIHTDAGGTAYRDSVKG (SEQ ID NO: 38). In certain embodiments, CDRH2 comprises the amino acid sequence of SIHTGGGGTAYRDSVKG (SEQ ID NO: 36). In certain embodiments, CDRH2 comprises the amino acid sequence of SIHTEAGGTAYRDSVKG (SEQ ID NO: 37). In certain embodiments, CDRH2 comprises the amino acid sequence of SIHTDAGGTAYRDSVKG (SEQ ID NO: 38). In certain embodiments, CDRH2 comprises the amino acid sequence of SIHTEGGGTAYRDSVKG (SEQ ID NO: 39).

In certain embodiments, CDRL1 comprises the amino acid sequence of KTSQGLVHSDGKTYFY (SEQ ID NO: 6) or KTSQGLVHSGGKTYFY (SEQ ID NO: 40). In certain embodiments, CDRL1 comprises the amino acid sequence of KTSQGLVHSDGKTYFY (SEQ ID NO: 6). In certain embodiments, CDRL1 comprises the amino acid sequence of KTSQGLVHSGGKTYFY (SEQ ID NO: 40).

In certain embodiments, CDRL3 comprises the amino acid sequence of AHGTYYPHT (SEQ ID NO: 14) or AQGTYYPHT (SEQ ID NO: 13). In certain embodiments, CDRL3 comprises the amino acid sequence of AHGTYYPHT (SEQ ID NO: 14). In certain embodiments, CDRL3 comprises the amino acid sequence of AQGTYYPHT (SEQ ID NO: 13).

In certain embodiments, the present disclosure provides an isolated antibody (eg, a humanized antibody) that specifically binds to ApoC3 (eg, human ApoC3), wherein the antibodies are SEQ ID NOs: 3, 36, respectively. , And 10; 3,37, and 10; 3,38, and 10; or VH domains comprising the amino acid sequences of CDRH1, CDRH2, and CDRH3 shown in 3, 39, and 10. In certain embodiments, the VH domain comprises the amino acid sequences of CDRH1, CDRH2, and CDRH3 set forth in SEQ ID NOs: 3, 36, and 10, respectively. In certain embodiments, the VH domain comprises the amino acid sequences of CDRH1, CDRH2, and CDRH3 set forth in SEQ ID NOs: 3, 37, and 10, respectively. In certain embodiments, the VH domain comprises the amino acid sequences of CDRH1, CDRH2, and CDRH3 set forth in SEQ ID NOs: 3, 38, and 10, respectively.

In certain embodiments, the present disclosure provides an isolated antibody that specifically binds to ApoC3 (eg, human ApoC3), wherein the antibodies are SEQ ID NOs: 6, 7, and 14; 40, 7, respectively. , And 14; or VL domains comprising the amino acid sequences of CDRL1, CDRL2, and CDRL3 shown in 40, 7, and 13. In certain embodiments, the VL domain comprises the amino acid sequences of CDRL1, CDRL2 and CDRL3 set forth in SEQ ID NOs: 6, 7 and 14, respectively. In certain embodiments, the VL domain comprises the amino acid sequences of CDRL1, CDRL2, and CDRL3 set forth in SEQ ID NOs: 40, 7 and 14, respectively.

In certain embodiments, the present disclosure provides an isolated antibody (eg, a humanized antibody) that specifically binds to ApoC3 (eg, human ApoC3), wherein the antibody is in the CDRH1, CDRH2, and CDRH3 regions. A heavy chain variable region comprising, and a light chain variable region comprising CDRL1, CDRL2, and CDRL3 regions, wherein the CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3 regions are, respectively, SEQ ID NOs: 3, 36, 10, 6, 7, and 14; 3, 37, 10, 40, 7, and 14; 3, 38, 10, 40, 7, and 14; 3, 39, 10, 6, 7, and 14; 3, 37, 10, 40, 7, and 13; or includes the amino acid sequences shown in 3, 38, 10, 40, 7, and 13. In certain embodiments, the CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3 regions contain the amino acid sequences set forth in SEQ ID NOs: 3, 36, 10, 6, 7, and 14, respectively. In certain embodiments, the CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3 regions contain the amino acid sequences set forth in SEQ ID NOs: 3, 37, 10, 40, 7, and 14, respectively. In certain embodiments, the CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3 regions contain the amino acid sequences set forth in SEQ ID NOs: 3, 38, 10, 40, 7, and 14, respectively.

In certain embodiments, the present disclosure is an isolated antibody (eg, a humanized antibody) that specifically binds to ApoC3 (eg, human ApoC3), with SEQ ID NOs: 42, 43, 44, 45, 46. , 47, 48, 49, 50, 51, 52, 53, 89, or 90 and at least 75%, 80%, 85%, 90%, 95%, or 100% (eg, at least 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99%) provides an isolated antibody comprising a heavy chain variable region comprising an amino acid sequence that is identical. .. In certain embodiments, the antibody is a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 89, or 90. including. In certain embodiments, the antibody comprises a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 42. In certain embodiments, the antibody comprises a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 43. In certain embodiments, the antibody comprises a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 44. In certain embodiments, the antibody comprises a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 45. In certain embodiments, the antibody comprises a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 46. In certain embodiments, the antibody comprises a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 47. In certain embodiments, the antibody comprises a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 48. In certain embodiments, the antibody comprises a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 49. In certain embodiments, the antibody comprises a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 50. In certain embodiments, the antibody comprises a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 51. In certain embodiments, the antibody comprises a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 52. In certain embodiments, the antibody comprises a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 53. In certain embodiments, the antibody comprises a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 89, optionally the heavy chain variable region being the amino acid set forth in SEQ ID NO: 15, 16, 17, or 18. Does not contain an array. In certain embodiments, the antibody comprises a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 90, optionally the heavy chain variable region being the amino acid set forth in SEQ ID NO: 15, 16, 17, or 18. Does not contain an array.

In certain embodiments, the present disclosure is an isolated antibody (eg, a humanized antibody) that specifically binds to ApoC3 (eg, human ApoC3), with SEQ ID NOs: 54, 55, 56, 57, 58. , 59, 60, 61, 62, 63, 64, 65, 91, or 92 and at least 75%, 80%, 85%, 90%, 95%, or 100% (eg, at least 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99%) provides an isolated antibody comprising a light chain variable region comprising an amino acid sequence that is identical. .. In certain embodiments, the antibody is a light chain variable region having the amino acid sequence set forth in SEQ ID NO: 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 91, or 92. including. In certain embodiments, the antibody comprises a light chain variable region having the amino acid sequence set forth in SEQ ID NO: 54. In certain embodiments, the antibody comprises a light chain variable region having the amino acid sequence set forth in SEQ ID NO: 55. In certain embodiments, the antibody comprises a light chain variable region having the amino acid sequence set forth in SEQ ID NO: 56. In certain embodiments, the antibody comprises a light chain variable region having the amino acid sequence set forth in SEQ ID NO: 57. In certain embodiments, the antibody comprises a light chain variable region having the amino acid sequence set forth in SEQ ID NO: 58. In certain embodiments, the antibody comprises a light chain variable region having the amino acid sequence set forth in SEQ ID NO: 59. In certain embodiments, the antibody comprises a light chain variable region having the amino acid sequence set forth in SEQ ID NO: 60. In certain embodiments, the antibody comprises a light chain variable region having the amino acid sequence set forth in SEQ ID NO: 61. In certain embodiments, the antibody comprises a light chain variable region having the amino acid sequence set forth in SEQ ID NO: 62. In certain embodiments, the antibody comprises a light chain variable region having the amino acid sequence set forth in SEQ ID NO: 63. In certain embodiments, the antibody comprises a light chain variable region having the amino acid sequence set forth in SEQ ID NO: 64. In certain embodiments, the antibody comprises a light chain variable region having the amino acid sequence set forth in SEQ ID NO: 65. In certain embodiments, the antibody comprises a light chain variable region having the amino acid sequence set forth in SEQ ID NO: 91. In certain embodiments, the antibody comprises a light chain variable region having the amino acid sequence set forth in SEQ ID NO: 92.

In certain embodiments, the present disclosure is an isolated antibody (eg, a humanized antibody) that specifically binds to ApoC3 (eg, human ApoC3), with SEQ ID NOs: 42, 43, 44, 45, 46. , 47, 48, 49, 50, 51, 52, 53, 89, or 90 and at least 75%, 80%, 85%, 90%, 95%, or 100% (eg, at least 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99%) Heavy chain variable regions containing identical amino acid sequences and SEQ ID NOs: 54, 55, 56, 57. , 58, 59, 60, 61, 62, 63, 64, 65, 91, or 92 and at least 75%, 80%, 85%, 90%, 95%, or 100% (eg, at least). 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99%) isolated, comprising a light chain variable region comprising an amino acid sequence that is identical. Provide antibodies. In certain embodiments, the antibody is a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 89, or 90. And a light chain variable region having the amino acid sequence set forth in SEQ ID NO: 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 91, or 92. In certain embodiments, the antibodies have SEQ ID NOs: 42 and 54, 43 and 55, 44 and 56, 45 and 57, 46 and 58, 46 and 54, 47 and 58, 47 and 54, 48 and 58, 48, respectively. And 54, 49 and 59, 49 and 60, 50 and 59, 50 and 60, 51 and 61, 52 and 62, 53 and 62, 43 and 63, 44 and 64, 45 and 65, 89 and 91, or 90 and It contains a heavy chain variable region and a light chain variable region having the amino acid sequence shown in 92. In certain embodiments, the antibody comprises a heavy chain variable region and a light chain variable region having the amino acid sequences set forth in SEQ ID NOs: 42 and 54, respectively. In certain embodiments, the antibody comprises a heavy chain variable region and a light chain variable region having the amino acid sequences set forth in SEQ ID NOs: 43 and 55, respectively. In certain embodiments, the antibody comprises a heavy chain variable region and a light chain variable region having the amino acid sequences set forth in SEQ ID NOs: 44 and 56, respectively. In certain embodiments, the antibody comprises a heavy chain variable region and a light chain variable region having the amino acid sequences set forth in SEQ ID NOs: 45 and 57, respectively. In certain embodiments, the antibody comprises a heavy chain variable region and a light chain variable region having the amino acid sequences set forth in SEQ ID NOs: 46 and 58, respectively. In certain embodiments, the antibody comprises a heavy chain variable region and a light chain variable region having the amino acid sequences set forth in SEQ ID NOs: 46 and 54, respectively. In certain embodiments, the antibody comprises a heavy chain variable region and a light chain variable region having the amino acid sequences set forth in SEQ ID NOs: 47 and 58, respectively. In certain embodiments, the antibody comprises a heavy chain variable region and a light chain variable region having the amino acid sequences set forth in SEQ ID NOs: 47 and 54, respectively. In certain embodiments, the antibody comprises a heavy chain variable region and a light chain variable region having the amino acid sequences set forth in SEQ ID NOs: 48 and 58, respectively. In certain embodiments, the antibody comprises a heavy chain variable region and a light chain variable region having the amino acid sequences set forth in SEQ ID NOs: 48 and 54, respectively. In certain embodiments, the antibody comprises a heavy chain variable region and a light chain variable region having the amino acid sequences set forth in SEQ ID NOs: 49 and 59, respectively. In certain embodiments, the antibody comprises a heavy chain variable region and a light chain variable region having the amino acid sequences set forth in SEQ ID NOs: 49 and 60, respectively. In certain embodiments, the antibody comprises a heavy chain variable region and a light chain variable region having the amino acid sequences set forth in SEQ ID NOs: 50 and 59, respectively. In certain embodiments, the antibody comprises a heavy chain variable region and a light chain variable region having the amino acid sequences set forth in SEQ ID NOs: 50 and 60, respectively. In certain embodiments, the antibody comprises a heavy chain variable region and a light chain variable region having the amino acid sequences set forth in SEQ ID NOs: 51 and 61, respectively. In certain embodiments, the antibody comprises a heavy chain variable region and a light chain variable region having the amino acid sequences set forth in SEQ ID NOs: 52 and 62, respectively. In certain embodiments, the antibody comprises a heavy chain variable region and a light chain variable region having the amino acid sequences set forth in SEQ ID NOs: 53 and 62, respectively. In certain embodiments, the antibody comprises a heavy chain variable region and a light chain variable region having the amino acid sequences set forth in SEQ ID NOs: 43 and 63, respectively. In certain embodiments, the antibody comprises a heavy chain variable region and a light chain variable region having the amino acid sequences set forth in SEQ ID NOs: 44 and 64, respectively. In certain embodiments, the antibody comprises a heavy chain variable region and a light chain variable region having the amino acid sequences set forth in SEQ ID NOs: 45 and 65, respectively. In certain embodiments, the antibody comprises a heavy chain variable region and a light chain variable region having the amino acid sequences set forth in SEQ ID NOs: 89 and 91, respectively, and optionally the heavy chain variable regions are SEQ ID NOs: 15 and 16. , 17, or 18 does not include the amino acid sequence shown. In certain embodiments, the antibody comprises a heavy chain variable region and a light chain variable region having the amino acid sequences set forth in SEQ ID NOs: 90 and 92, respectively, and optionally the heavy chain variable regions are SEQ ID NOs: 15 and 16. , 17, or 18 does not include the amino acid sequence shown.

In certain embodiments, the present disclosure provides an isolated antibody that specifically binds to ApoC3 (eg, human ApoC3), the antibody being a heavy chain having complementarity determining regions CDRH1, CDRH2, and CDRH3. It comprises variable regions and light chain variable regions having complementarity determining regions CDRL1, CDRL2, and CDRL3.
(A) CDRH1 comprises the amino acid sequence of TYSMR (SEQ ID NO: 3).
(B) CDRH 2 comprises the amino acid sequence of SIX ₁ TDGGGTAYRDSVKG (where X ₁ is S or H in the formula) (SEQ ID NO: 4).
(C) CDRH3 comprises the amino acid sequence of X ₂ GYSD (where X ₂ is A or H in the formula) (SEQ ID NO: 5).
(D) CDRL1 comprises the amino acid sequence of KTSQGLVHSDGKTYFY (SEQ ID NO: 6).
(E) CDRL2 comprises the amino acid sequence of QVSNRAS (SEQ ID NO: 7) and / or (f) CDRL3 is AX _{3 GTYYPHT} (where X3 is Q or H in the formula) (SEQ ID NO: 8). Includes amino acid sequence.

In certain embodiments, the present disclosure provides an isolated antibody that specifically binds to ApoC3 (eg, human ApoC3), the antibody being a heavy chain having complementarity determining regions CDRH1, CDRH2, and CDRH3. It comprises variable regions and light chain variable regions having complementarity determining regions CDRL1, CDRL2, and CDRL3.
(A) CDRH1 comprises the amino acid sequence of TYSMR (SEQ ID NO: 3).
(B) CDRH 2 comprises the amino acid sequence of SIX ₁ TDGGGTAYRDSVKG (where X ₁ is S or H in the formula) (SEQ ID NO: 4).
(C) CDRH3 comprises the amino acid sequence of X ₂ GYSD (where X ₂ is A or H in the formula) (SEQ ID NO: 5).
(D) CDRL1 comprises the amino acid sequence of KTSQGLVHSDGKTYFY (SEQ ID NO: 6).
(E) CDRL2 comprises the amino acid sequence of QVSNRAS (SEQ ID NO: 7), and (f) CDRL3 is the amino acid sequence of AX _{3 GTYYPHT} (where X3 is Q or H in the formula) (SEQ ID NO: 8). Including
In the formula, at least one of X ₁ , X ₂ , and X ₃ is H.

In certain embodiments, the present disclosure provides an isolated antibody that specifically binds to ApoC3 (eg, human ApoC3).
(A) CDRH1, which comprises the amino acid sequence of SEQ ID NO: 3.
(B) CDRH2, which comprises the amino acid sequence of SEQ ID NO: 9 or 11.
(C) CDRH3, which comprises the amino acid sequence of SEQ ID NO: 10 or 12.
(D) CDRL1, which comprises the amino acid sequence of SEQ ID NO: 6.
(E) CDRL2 comprising the amino acid sequence of SEQ ID NO: 7 and / or (f) CDRL3 comprising the amino acid sequence of SEQ ID NO: 13 or 14.

In certain embodiments, the present disclosure provides an isolated antibody that specifically binds to ApoC3 (eg, human ApoC3).
(A) CDRH1, which comprises the amino acid sequence of SEQ ID NO: 3.
(B) CDRH2, which comprises the amino acid sequence of SEQ ID NO: 9 or 11.
(C) CDRH3, which comprises the amino acid sequence of SEQ ID NO: 10 or 12.
(D) CDRL1, which comprises the amino acid sequence of SEQ ID NO: 6.
(E) CDRL2 comprising the amino acid sequence of SEQ ID NO: 7 and (f) CDRL3 comprising the amino acid sequence of SEQ ID NO: 13 or 14
The isolated antibodies do not contain the CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3 sequences set forth in SEQ ID NOs: 3, 9, 10, 6, 7, and 13, respectively.

In certain embodiments, the present disclosure provides an isolated antibody that specifically binds to ApoC3 (eg, human ApoC3), wherein the antibodies are SEQ ID NOs: 3, 9, and 10; 3, 11, respectively. , And 10; 3, 9, and 12; or VH domains comprising the amino acid sequences of CDRH1, CDRH2, and CDRH3 shown in 3, 11, and 12. In certain embodiments, the VH domain comprises the amino acid sequences of CDRH1, CDRH2, and CDRH3 set forth in SEQ ID NOs: 3, 11 and 10, respectively. In certain embodiments, the VH domain comprises the amino acid sequences of CDRH1, CDRH2, and CDRH3 set forth in SEQ ID NOs: 3, 9, and 12, respectively. In certain embodiments, the VH domain comprises the amino acid sequences of CDRH1, CDRH2, and CDRH3 set forth in SEQ ID NOs: 3, 11 and 12, respectively.

In certain embodiments, the present disclosure provides an isolated antibody that specifically binds to ApoC3 (eg, human ApoC3), wherein the antibodies are SEQ ID NOs: 6, 7, and 13; or 6, respectively. Includes a VL domain comprising the amino acid sequences of CDRL1, CDRL2, and CDRL3 shown in 7 and 14. In certain embodiments, the VL domain comprises the amino acid sequences of CDRL1, CDRL2, and CDRL3 set forth in SEQ ID NOs: 6, 7, and 14, respectively.

In certain embodiments, the present disclosure provides an isolated antibody that specifically binds to ApoC3 (eg, human ApoC3), wherein the antibody comprises a heavy chain variable region comprising the CDRH1, CDRH2, and CDRH3 regions. , CDRL1, CDRL2, and a light chain variable region comprising the CDRL3 region, wherein the CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3 regions are SEQ ID NOs: 3, 11, 10, 6, 7, and, respectively. 13; 3, 9, 12, 6, 7, and 13; 3, 9, 10, 6, 7, and 14; 3, 11, 10, 6, 7, and 14; 3, 9, 12, 6, 7 , And 14; or 3, 11, 12, 6, 7, and 13; or include the amino acid sequences set forth in 3, 11, 12, 6, 7, and 13. In certain embodiments, the CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3 regions contain the amino acid sequences set forth in SEQ ID NOs: 3, 11, 10, 6, 7, and 14, respectively. In certain embodiments, the CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3 regions contain the amino acid sequences set forth in SEQ ID NOs: 3, 9, 12, 6, 7, and 14, respectively.

In certain embodiments, the present disclosure is an isolated antibody that specifically binds to ApoC3 (eg, human ApoC3) and is at least 75 with the amino acid sequence set forth in SEQ ID NO: 15, 16, 17, or 18. %, 80%, 85%, 90%, 95%, or 100% (eg, at least 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99%). ) Provide an isolated antibody comprising a heavy chain variable region containing an identical amino acid sequence. In certain embodiments, the antibody comprises a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 15, 16, 17, or 18. In certain embodiments, the antibody comprises a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 16. In certain embodiments, the antibody comprises a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 17. In certain embodiments, the antibody comprises a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 18.

In certain embodiments, the present disclosure is an isolated antibody that specifically binds to ApoC3 (eg, human ApoC3), at least 75%, 80%, with the amino acid sequence set forth in SEQ ID NO: 19 or 20. Amino acids that are 85%, 90%, 95%, or 100% (eg, at least 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99%) identical An isolated antibody comprising a light chain variable region comprising a sequence is provided. In certain embodiments, the antibody comprises a light chain variable region having the amino acid sequence set forth in SEQ ID NO: 20.

In certain embodiments, the present disclosure is an isolated antibody that specifically binds to ApoC3 (eg, human ApoC3) and is at least 75 with the amino acid sequence set forth in SEQ ID NO: 15, 16, 17, or 18. %, 80%, 85%, 90%, 95%, or 100% (eg, at least 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99%). ) At least 75%, 80%, 85%, 90%, 95%, or 100% (eg, at least 86,) of the heavy chain variable region comprising the same amino acid sequence and the amino acid sequence set forth in SEQ ID NO: 19 or 20. 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99%) Isolated antibody comprising a light chain variable region comprising an amino acid sequence that is identical. offer. In certain embodiments, the antibody comprises a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 15, 16, 17, or 18, and a light chain variable region having the amino acid sequence set forth in SEQ ID NO: 19 or 20. including. In certain embodiments, the antibody is a heavy chain having the amino acid sequences set forth in SEQ ID NOs: 16 and 19, 17 and 19, 18 and 19, 15 and 20, 16 and 20, 17 and 20, or 18 and 20, respectively. Includes variable region and light chain variable region. In certain embodiments, the antibody comprises a heavy chain variable region and a light chain variable region having the amino acid sequences set forth in SEQ ID NOs: 16 and 20, respectively. In certain embodiments, the antibody comprises a heavy chain variable region and a light chain variable region having the amino acid sequences set forth in SEQ ID NOs: 17 and 20, respectively.

Any Ig constant region can be used in the isolated antibody disclosed herein. In certain embodiments, the Ig constant region is a constant region of a human IgG, IgE, IgM, IgD, IgA, or IgY immunoglobulin (Ig) molecule and / or any class of immunoglobulin molecule (eg, IgG). ₁ , IgG ₂ , IgG ₃ , IgG ₄ , IgA ₁ , and IgA ₂ ), or a constant region of any subclass (eg, IgG _2a and IgG _2b ). In certain embodiments, the Ig constant region is a human or humanized Ig constant region.

In certain embodiments, the constant region is a variant of the wild-type human Ig (eg, IgG) heavy chain constant region, and the variant human Ig heavy chain constant region corresponds to the corresponding wild to human FcRn under the same conditions. Increased at acidic pH (eg, pH 5.5-pH 6) to human neonatal Fc receptors (FcRn) compared to the affinity of the type human Ig heavy chain constant region (eg, at least 1.5, 2, 2). .5, 3, 4, 5, 6, 7, 8, 9, 10, 15, or 20-fold increased) Affinity. In certain embodiments, the variant human Ig heavy chain constant region is relative to the human neonatal Fc receptor (FcRn) as compared to the affinity of the wild-type human Ig heavy chain constant region for human FcRn under the same conditions. Similar or decreased (eg, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1. Has an equivalent or reduced affinity of 8, 1.9, or less than 2-fold. In certain embodiments, the constant region is one or more amino acids (eg, 1) from a wild-type Ig (eg, IgG) constant domain or an FcRn binding fragment thereof (eg, Fc or hinge-Fc domain fragment). Has one or more substitutions, insertions, or deletions). In certain embodiments, the half-life of an antibody having a variant constant region in vivo is increased as compared to the half-life of a corresponding antibody having a wild-type constant domain or FcRn-binding fragment thereof in vivo. For example, with respect to examples of mutations that increase the half-life of an antibody in vivo, WO 02/060919, WO 98/23289, and WO 97/34631, and US Pat. No. 5,869,046. , No. 6,121,022, No. 6,277,375, No. 6,165,745, No. 8,088,376, and No. 8,163,881. All of these are incorporated herein by reference in their entirety. In certain embodiments, one or more different amino acids are numbered according to the EU numbering system, a second constant (CH2) domain (residues 231 to 340 of human IgG1), and / or _a third. It is _a constant (CH3) domain (residues 341 to 447 of human IgG1). In certain embodiments, the constant regions of IgG (eg, IgG ₁ , IgG ₂ , or IgG ₄ ) of the antibodies disclosed herein are numbered according to the EU numbering system, 252, 254, respectively. And contains the amino acids tyrosine (Y), threonine (T), and glutamic acid (E) at position 256. See U.S. Pat. No. 7,658,921, which is incorporated herein by reference in its entirety. This IgG type, referred to as "YTE IgG", has been shown to display a 4-fold increased half-life compared to the wild-type version of the same antibody (Dall'Acqua WF et al., (Dall'Acqua WF et al.,). 2006) See J Biol Chem 281: 23514-24, which is incorporated herein by reference in its entirety). In certain embodiments, the constant regions of the IgG (eg, IgG ₁ ) of the antibodies disclosed herein are the amino acids alanine (A), serine (S) at position 434, numbered according to the EU numbering system. , Tyrosine (Y), or phenylalanine (F). In certain embodiments, the IgG constant regions of the antibodies disclosed herein (eg, IgG ₁ , IgG ₂ , or IgG ₄ ) are numbered according to the EU numbering system, respectively, 433. It contains the amino acids lysine (K), phenylalanine (F), and tyrosine (Y) at positions 434 and 436. In certain embodiments, the constant regions of IgG (eg, IgG ₁ , IgG ₂ , or IgG ₄ ) of the antibodies disclosed herein are numbered according to the EU numbering system, at positions 428 and 434, respectively. Contains the amino acids leucine (L) and serine (S). Additional IgG constant regions that may have increased affinity for FcRn under acidic conditions are described in Ward et al. , Mol. Immunol. (2015) 67 (200): 131-41, which is incorporated herein by reference in its entirety. In certain embodiments, the antibody is one of amino acid residues at positions 251-257, 285-290, 308-314, 385-389, and 428-436, numbered according to the EU numbering system. Contains an IgG constant domain containing two or more amino acid substitutions. In certain embodiments, the isolated antibodies disclosed herein are SEQ ID NOs: 21, 22, 23, 24, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, Includes a heavy chain constant region comprising the amino acid sequence set forth in 85, or 86. In certain embodiments, the isolated antibody disclosed herein comprises a light chain constant region comprising the amino acid sequence set forth in SEQ ID NO: 25 or 26.

In certain embodiments, the present disclosure, an isolated antibody that specifically binds to ApoC3 (eg, human ApoC3), to SEQ ID NOs: 66, 67, 68, 69, 70, 71, 72, or 73. Provided is an isolated antibody comprising a heavy chain containing the indicated amino acid sequence. In certain embodiments, the present disclosure is an isolated antibody that specifically binds to ApoC3 (eg, human ApoC3) and comprises a light chain comprising the amino acid sequence set forth in SEQ ID NO: 74. Provide an antibody. In certain embodiments, the present disclosure is an isolated antibody that specifically binds to ApoC3 (eg, human ApoC3), with SEQ ID NOs: 66 and 74, 67 and 74, 68 and 74, 69, respectively. Provided are isolated antibodies comprising heavy and light chains comprising the amino acid sequences set forth in 74, 70 and 74, 71 and 74, 72 and 74, or 73 and 74.

In certain embodiments, the present disclosure is an isolated antibody that specifically binds to ApoC3 (eg, human ApoC3) with SEQ ID NOs: 27, 28, 29, 30, 31, 32, 33, or 34. Provided is an isolated antibody comprising a heavy chain comprising the amino acid sequence shown in. In certain embodiments, the present disclosure comprises an isolated antibody that specifically binds to ApoC3 (eg, human ApoC3) and comprises a light chain comprising the amino acid sequence set forth in SEQ ID NO: 35. Provide the antibody. In certain embodiments, the present disclosure is an isolated antibody that specifically binds to ApoC3 (eg, human ApoC3), with SEQ ID NOs: 27 and 35, 28 and 35, 29 and 35, 30, respectively. 35, 31 and 35, 32 and 35, 33 and 35, or isolated antibodies comprising heavy and light chains comprising the amino acid sequences set forth in 34 and 35 are provided.

3. 3. Usage ApoC3 inhibits hepatocyte uptake and clearance of TRL (eg, VLDL) and TRL remnants, thereby inhibiting lipoprotein lipase-mediated lipolysis of TRL (eg, VLDL) in the blood of the subject. It acts to increase triglyceride levels. In certain embodiments, the anti-ApoC3 antibodies disclosed herein reduce the ability of ApoC3 to inhibit TRL (eg, VLDL) and TRL remnant uptake and clearance by hepatocytes, or TRL (eg, VLDL). ) Can weaken the ability of ApoC3 to inhibit lipoprotein lipase-mediated lipolysis. Accordingly, in certain embodiments, the present disclosure provides a method for inhibiting the activity of ApoC3 in the blood of a subject, which method presents the subject with an effective amount of an anti-ApoC3 antibody or anti-ApoC3 antibody disclosed herein. Includes administration of the pharmaceutical composition. In certain embodiments, the activity of ApoC3 is inhibition of TRL (eg, VLDL) and TRL remnant uptake and clearance by hepatocytes. In certain embodiments, the activity of ApoC3 is inhibition of lipoprotein lipase-mediated lipolysis of TRL. In certain embodiments, the activity of ApoC3 is inhibition of hepatocyte uptake and clearance of TRL (eg, VLDL) and TRL remnants, as well as inhibition of lipoprotein lipase-mediated lipolysis of TRL.

The anti-ApoC3 antibodies disclosed herein are useful for increasing the rate of clearance of ApoC3 and / or ApoB (eg, ApoB48 and / or ApoB100) from the blood of interest. Accordingly, in certain embodiments, the present disclosure provides a method for increasing the rate of clearance of ApoC3 and / or ApoB (eg, ApoB48 and / or ApoB100) from a subject's blood, the method of which is the subject. Includes administration of an effective amount of the anti-ApoC3 antibody or pharmaceutical composition disclosed herein.

The anti-ApoC3 antibodies disclosed herein are useful for reducing the levels of ApoC3 and / or ApoB (eg, ApoB48 and / or ApoB100) in the blood of a subject. Accordingly, in certain embodiments, the present disclosure provides a method for reducing the levels of ApoC3 and / or ApoB (eg, ApoB48 and / or ApoB100) in the blood of a subject, the method comprising the subject. It comprises administering an effective amount of the anti-ApoC3 antibody or pharmaceutical composition disclosed herein. In certain embodiments, the method is ApoC3 and / or ApoB (eg, ApoB48 and / or ApoB100) in the blood of a subject as assessed by the methods disclosed herein or by methods known to those of skill in the art. ) Levels of at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, Reduce by 80%, 85%, 90%, 95%, 98%, or 99%. In certain embodiments, the method is ApoC3 and / or ApoB (eg, ApoB48 and / or ApoB100) in the blood of subject as assessed by the methods disclosed herein or by methods known to those of skill in the art. ) Levels at least about 1.1 times, 1.2 times, 1.3 times, 1.4 times, 1.5 times, 2 times, 2.5 times, 3 times, 3.5 times, 4 times, 4.5 times, 5 times, 6 times, 7 times, 8 times, 9 times, 10 times, 15 times, 20 times, 30 times, 40 times, 50 times, 60 times, 70 times, 80 times, 90 times, Or reduce it 100 times. In certain embodiments, the reduction in levels of ApoC3 and / or ApoB (eg, ApoB48 and / or ApoB100) in the blood of a subject is at least 1, 2, 3, 4, 5, 6, 7, 8, 9, ,. It is maintained for 10, 15, 20, 25, 30, 35, 40, 45, or 50 days, or at least 1, 2, 3, 4, 5, 6, 7, or 8 weeks.

The anti-ApoC3 antibodies disclosed herein are useful for reducing triglyceride levels in the blood of a subject. Accordingly, in certain embodiments, the present disclosure provides a method for reducing triglyceride levels in a subject's blood, wherein the subject is given an effective amount of an anti-ApoC3 antibody or anti-ApoC3 antibody disclosed herein. Includes administration of a pharmaceutical composition.

The anti-ApoC3 antibodies disclosed herein are useful for the treatment of hypertriglyceridemia. Accordingly, in certain embodiments, the present disclosure provides a method for treating hypertriglyceridemia in a subject, which method presents the subject in an effective amount of an anti-ApoC3 antibody or pharmaceutically disclosed herein. Includes administration of the composition. In certain embodiments, the present disclosure provides a method for treating chylomicronemia in a subject, which method presents the subject in an effective amount of an anti-ApoC3 antibody or pharmaceutical composition disclosed herein. Includes administration of. In certain embodiments, the present disclosure provides a method for treating chylomicronemia syndrome in a subject, the method of which is an effective amount of an anti-ApoC3 antibody or pharmaceutical composition disclosed herein. Includes administration of material.

The anti-ApoC3 antibodies disclosed herein are useful for the treatment and prevention of postprandial lipoemia in a subject. Accordingly, in certain embodiments, the present disclosure provides a method for inhibiting postprandial lipoemia in a subject, which method presents the subject in an effective amount of an anti-ApoC3 antibody or pharmaceutically disclosed herein. Includes administration of the composition. Anti-ApoC3 antibodies can be administered to a subject before, during, or after a meal.

Although not bound by theory, Applicants, in certain embodiments, if the antibodies disclosed herein are administered to a subject before, during, or after a meal. It is believed that postprandial chylomicrons or chylomicrons remnant levels in the subject can be reduced. Accordingly, in certain embodiments, the present disclosure provides a method for reducing postprandial chylomicrons or chylomicrons remnant levels in a subject, which method presents the subject in an effective amount of anti-antibody disclosed herein. Includes administration of ApoC3 antibody or pharmaceutical composition. Anti-ApoC3 antibodies can be administered to a subject before, during, or after a meal.

Lowering blood triglyceride levels in patients with hypertriglyceridemia may reduce the risk of developing pancreatitis. Accordingly, in certain embodiments, the present disclosure provides a method for reducing the risk of pancreatitis in a subject with hypertriglyceridemia, the method being disclosed to the subject in an effective amount herein. Includes administration of an anti-ApoC3 antibody or pharmaceutical composition.

The anti-ApoC3 antibodies disclosed herein are useful for reducing the risk of cardiovascular disease in a subject. Accordingly, in certain embodiments, the present disclosure provides a method for reducing the risk of cardiovascular disease in a subject with hypertriglyceridemia, the method being disclosed to the subject in an effective amount herein. Includes administration of anti-ApoC3 antibody or pharmaceutical composition. The risk of developing any cardiovascular disease associated with or caused by hypertriglyceridemia or excess postprandial lipoemia is reduced by administration of the anti-ApoC3 antibody or pharmaceutical composition disclosed herein. obtain. Cardiovascular diseases that may reduce the risk include, but are not limited to, coronary artery disease, atherosclerosis, angina, myocardial infarction, and stroke.

The anti-ApoC3 antibody or pharmaceutical composition disclosed herein can be administered either alone or in combination with additional therapeutic agents. In certain embodiments, the additional therapeutic agent is another lipid lowering agent. Any one or more lipid lowering agents can be used in combination with the anti-ApoC3 antibody or pharmaceutical composition disclosed herein. Suitable lipid-lowering agents include HMG-CoA reductase inhibitors (eg, atorvastatin, fluvastatin, robastatin, pitabastatin, pravastatin, rosbatatin, or simbatatin), fibrato, nicotinic acid, bile acid sequestering agents (eg, cholestiramine, cholestipol). , And cholesterol), dietary cholesterol absorption inhibitors (eg, ezetimib), microsomal triglyceride transport protein (MTP) inhibitors (eg, lomitapide), phytosterol, pancreatic lipase inhibitors (eg, orlistat), cholesteryl ester transfer protein inhibitors , Squalene synthase inhibitors (eg, TAK-475, zaragodic acid, and RPR 107393), ApoA-1 Milano, succinobcol (AGI-1067), apoprotein B inhibitors (eg, mipomersen), and proprotein conversion. Includes, but is not limited to, inhibitors of the enzyme subtilicin / kexin type 9 (PCSK9), such as, but not limited to, allylomab, evolocumab, and bocosizumab. In certain embodiments, the additional lipid-lowering agent is a combination of ezetimibe and an HMG-CoA reductase inhibitor. In certain embodiments, the lipid-lowering agent is a combination of ezetimibe, an HMG-CoA reductase inhibitor, and a PCSK9 inhibitor.

The anti-ApoC3 antibody or pharmaceutical composition disclosed herein can be delivered to a subject by a variety of routes. These include, but are not limited to, parenteral, intradermal, intramuscular, intraperitoneal, intravenous, and subcutaneous routes. In certain embodiments, the antibodies or pharmaceutical compositions disclosed herein are delivered subcutaneously or intravenously.

The amount of anti-ApoC3 or pharmaceutical composition disclosed herein that will be effective in treating or preventing a disease will depend on the nature of the disease and will be empirically determined by standard clinical techniques. can do. The exact dose used for the composition will also depend on the route of administration and the severity of the infection or disease caused by it and should be determined according to the judgment of the practitioner and the circumstances of each subject. For example, the effective dose may also be the means of administration, the target site, the patient's physiological condition (including age, weight, and health), whether the patient is human or animal, other drugs administered, or treatment. Can vary depending on whether is prophylactic or therapeutic. The anti-ApoC3 antibody or pharmaceutical composition disclosed herein is administered at any frequency (eg, approximately every week, every two weeks, every three weeks, every four weeks, every one month, or every two months). can do. Patients are usually human, but non-human mammals, including transgenic mammals, can also be treated. Therapeutic dosages and regimens are optimally titrated to optimize safety and efficacy.

The anti-ApoC3 antibodies disclosed herein also include classical immunohistochemistry known to those of skill in the art, including immunoassays such as enzyme-linked immunosorbent assay (ELISA), immunoprecipitation, or Western blotting. It can also be used to assay ApoC3 (eg, human ApoC3) protein levels in a biological sample using a specific method. Suitable assay labels are known in the art and include enzyme labels such as glucose oxidase, iodine ( ¹²⁵ I, ¹²¹ I), carbon ( ¹⁴ C), sulfur ( ³⁵ S), tritium ( ³ ). H), radioactive isotopes such as indium ( ¹²¹ In), and technetium ( ⁹⁹ Tc), luminescent labels such as luminol, fluorescent labels such as fluorescein and rhodamine, and biotin. Such labels can be used to label the antibodies disclosed herein. Alternatively, a secondary antibody that recognizes the anti-ApoC3 antibody disclosed herein can be labeled and used in combination with the anti-ApoC3 antibody to detect ApoC3 (eg, human ApoC3) protein levels. be able to.

Assaying for ApoC3 (eg, human ApoC3) protein expression levels can be direct (eg, by determining or predicting absolute protein levels) or relatively (eg, disease in a second biological sample). It is intended to include qualitatively or quantitatively measuring or predicting the level of ApoC3 (eg, human ApoC3) protein in a first biological sample, either by comparison with related protein levels). .. The expression level of the ApoC3 (eg, human ApoC3) polypeptide in the first biological sample can be measured or estimated and compared to standard ApoC3 (eg, human ApoC3) protein levels, the standard having the disease. It is determined by averaging the levels of a population of individuals who do not have the disease or are obtained from a second biological sample obtained from an individual who does not. As will be understood in the art, once the "standard" ApoC3 (eg, human ApoC3) polypeptide level is known, it can be used repeatedly as a standard for comparison.

As used herein, the term "biological sample" refers to any living body obtained from a subject, cell line, tissue, or other cell source that may express ApoC3 (eg, human ApoC3). Refers to a sample. Methods for obtaining tissue biopsies and body fluids from animals (eg, humans) are well known in the art. Biological samples include peripheral blood mononuclear cells.

The anti-ApoC3 antibodies disclosed herein can be used for prognostic, diagnostic, monitoring, and screening applications, which are well known and standard to those of skill in the art, and in vitro according to the present specification. And in vivo applications are included. Prognosis for in vitro assessment and assessment of immune system status or immune response Prognosis, diagnosis, monitoring, and screening assays and kits are used to predict, diagnose, and monitor and are known to have elevated ApoC3 activity. Patient samples can be assessed, including those that are or are suspected of having it. In one embodiment, the anti-ApoC3 antibody can be used for immunohistochemical testing of biopsy samples. In another embodiment, anti-ApoC3 antibodies can be used to detect ApoC3 (eg, human ApoC3) levels that can lead to certain disease symptoms. The anti-ApoC3 antibody disclosed herein may carry a detectable label or functional label. When fluorescent labels are used, specific binding members can be obtained using a combination of microscopic and oral fluorescently labeled cell preparative analysis (FACS) currently available, or both method procedures known in the art. Can be identified and quantified. The anti-ApoC3 antibody disclosed herein may carry a fluorescent label. Exemplary fluorescent labels include, for example, reactive and conjugated probes such as aminocoumarin, fluorescein, and Texas Red, Alexa Fluor dyes, Cy dyes, and DyLight dyes. Anti-ApoC3 antibodies are isotopes ³ H, ¹⁴ C, ³² P, ³⁵ S, ³⁶ Cl, ⁵¹ Cr, ⁵⁷ Co, ⁵⁸ Co, ⁵⁹ Fe, ⁶⁷ Cu, ⁹⁰ Y, ⁹⁹ Tc, ¹¹¹ In, ¹¹⁷ Lu, ¹²¹ . It can carry radioactive labels such as I, ¹²⁴ I, ¹²⁵ I, ¹³¹ I, ¹⁹⁸ Au, ²¹¹ At, ²¹³ Bi, ²²⁵ Ac, and ¹⁸⁶ Re. When radioactive labels are used, currently available counting procedures known in the art can be used to identify and quantify specific binding of anti-ApoC3 antibodies to ApoC3 (eg, human ApoC3). can. If the label is an enzyme, detection is achieved by any of the currently used colorimetric, spectrophotometric, fluorescent spectrophotometric, current measurement, or gas quantification techniques known in the art. Can be done. This can be achieved by contacting the sample or control sample with the anti-ApoC3 antibody under conditions that allow the formation of a complex of the antibody with ApoC3 (eg, human ApoC3). Any complex formed between the antibody and ApoC3 (eg, human ApoC3) is detected and compared in the sample and control. The antibodies disclosed herein can also be used to purify ApoC3 (eg, human ApoC3) via immunoaffinity purification. Also included herein are assay systems that can be prepared in the form of test kits for quantitative analysis of the extent of the presence of ApoC3 (eg, human ApoC3), for example. The system or test kit may include a labeled component, eg, a labeled ApoC3 antibody, and one or more additional immunochemical reagents.

4. Pharmaceutical Compositions A pharmaceutical composition comprising an anti-ApoC3 antibody disclosed herein having the desired purity in a pharmaceutically acceptable carrier, excipient, or stabilizer is provided herein. (Remington's Pharmaceutical Sciences (1990) Mac Publishing Co., Easton, PA). Acceptable carriers, excipients, or stabilizers are non-toxic to the recipient at the dosage and concentration used, such as buffers such as phosphates, citric acids, and other organic acids; Excipients containing ascorbic acid and methionine; preservatives (octadecyldimethylbenzylammonium chloride; hexamethonium chloride; benzalconium chloride, benzethonium chloride; phenol, butyl, or benzyl alcohol; alkylparabens such as methyl or propylparaben; catechol Resolsinol; Cyclohexanol; 3-Pentanol; and m-cresol etc.); Low molecular weight (less than about 10 residues) polypeptides; Proteins such as serum albumin, gelatin, or immunoglobulins; Hydrophilic polymers such as polyvinylpyrrolidone; Amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrin; chelating agents such as EDTA; sucrose, mannitol, trehalose, or sorbitol, etc. Sugars; salt-forming counterions such as sodium; metal complexes (eg, Zn-protein complexes); or nonionic surfactants such as TWEEN®, PLURONICS®, or polyethylene glycol (PEG). included.

In certain embodiments, the pharmaceutical composition comprises, in a pharmaceutically acceptable carrier, the anti-ApoC3 antibody disclosed herein and optionally one or more additional prophylactic or therapeutic agents. .. In certain embodiments, the pharmaceutical composition comprises an effective amount of an antibody disclosed herein and optionally one or more additional prophylactic or therapeutic agents in a pharmaceutically acceptable carrier. include. In some embodiments, the antibody is the only active ingredient contained in the pharmaceutical composition. The pharmaceutical compositions disclosed herein may be useful for inhibiting ApoC3 activity and treating diseases such as cancer or infections.

Pharmaceutically acceptable carriers used in parenteral preparations include aqueous vehicles, non-aqueous vehicles, antibacterial agents, tonicity agents, buffers, antioxidants, local anesthetics, suspending agents and dispersions. Includes agents, emulsifiers, sequestering or chelating agents, as well as other pharmaceutically acceptable substances. Examples of aqueous vehicles include sodium chloride injection, Ringer's solution, isotonic dextrose injection, sterile water injection, dextrose and lactated Ringer's solution. Non-aqueous oral vehicles include plant-derived non-volatile oils, cottonseed oil, corn oil, sesame oil, and peanut oil. Bacteriostatic or bacteriostatic concentrations of antimicrobial agent may be added to the oral preparation packaged in a multi-dose container, including phenol or cresol, mercury, benzyl alcohol, chlorobutanol, methyl and propyl. Includes p-hydroxybenzoic acid esters, thimerosal, benzalkonium chloride, and benzethonium chloride. Isotonic agents include sodium chloride and dextrose. The buffer contains phosphoric acid and citric acid. Antioxidants include sodium bisulfate. Local anesthetics include procaine hydrochloride. Suspensions and dispersants include sodium carboxymethylcellulouse, hydroxypropylmethylcellulose, and polyvinylpyrrolidone. The emulsifier includes Polysorbate 80 (TWEEN® 80). Metal ion sequestering or chelating agents include EDTA. Pharmaceutical carriers also include ethyl alcohol, polyethylene glycol, and propylene glycol for water-miscible vehicles, as well as sodium hydroxide, hydrochloric acid, citric acid, or lactic acid for pH control.

The pharmaceutical composition can be formulated for any route of administration to the subject. Specific examples of routes of administration include intranasal, oral, lung, transdermal, intradermal, and parenteral. Parenteral administration, characterized by either subcutaneous, intramuscular, or intravenous injection, is also contemplated herein. Injections can be prepared in either conventional form as a liquid solution or suspension, in solid form suitable for dissolution or suspension in liquid prior to injection, or as an emulsion. Injections, solutions, and emulsions also contain one or more excipients. Suitable excipients are, for example, water, saline, dextrose, glycerol, or ethanol. In addition, if desired, the pharmaceutical composition administered may be a small amount of a non-toxic auxiliary substance, such as a wetting agent or emulsifier, a pH buffer, a stabilizer, a solubility enhancer, and, for example, sodium acetate, sorbitan monolaurate. , Triethanolamine oleate, and other such agents such as cyclodextrin may also be included.

Preparations for parenteral administration of the antibody include sterile solutions for injection, dry sterile soluble products (including subcutaneous tablets) such as freeze-dried powders to be combined with the solvent immediately before use, and sterile for injection. Includes suspensions, dry sterile insoluble products for immediate vehicle use, and sterile emulsions. The solution may be either aqueous or non-aqueous.

When administered intravenously, suitable carriers include saline or phosphate buffered saline (PBS), and thickeners and solubilizers such as glucose, polyethylene glycol, and polypropylene glycol, and mixtures thereof. Contains agents.

Local mixtures containing antibodies are prepared as described for topical and systemic administration. The resulting mixture can be a solution, suspension, emulsion, etc., cream, gel, ointment, emulsion, solution, elixir, lotion, suspension, tincture, paste, foam, etc. It can be formulated as an aerosol, a lotion, a spray, a suppository, a bandage, a skin patch, or any other formulation suitable for topical administration.

The anti-ApoC3 antibodies disclosed herein can be formulated as an aerosol, eg, for topical administration by inhalation (eg, an aerosol for delivery of steroids useful in the treatment of inflammatory diseases, especially asthma. , US Pat. Nos. 4,044,126, 4,414,209, and 4,364,923). These formulations for respiratory administration can be in the form of aerosols or solutions for nebulizers, alone or in combination with an inert carrier such as lactose, or micropowdered agents for blowing. In such cases, the particles of the pharmaceutical product will have a diameter of less than 50 microns in one embodiment and less than 10 microns in one embodiment.

The anti-ApoC3 antibodies disclosed herein are in the form of gels, creams, and lotions for local or topical administration, eg, for topical application to mucous membranes such as in the skin and in the eye. , And can be formulated for application in the eye or in the cistern or in the spinal cord. Topical administration is intended for transdermal delivery and also for ocular or mucosal administration or inhalation therapy. Nasal drops may also be administered with the antibody alone or in combination with other pharmaceutically acceptable excipients.

Transdermal patches, including ion shaking devices and electrophoresis devices, are well known in the art and can be used to administer antibodies. For example, such patches include US Pat. Nos. 6,267,983, 6,261,595, 6,256,533, 6,167,301, 6,024,975. Disclosed in Nos. 6,010,715, 5,985,317, 5,983,134, 5,948,433, and 5,860,957. ing.

In certain embodiments, pharmaceutical compositions, including those disclosed herein, are lyophilized powders that can be reconstituted for administration as solutions, emulsions, and other mixtures. .. It can also be reconstituted and formulated as a solid or gel. The lyophilized powder is prepared by dissolving the antibodies disclosed herein or pharmaceutically acceptable derivatives thereof in a suitable solvent. In some embodiments, the lyophilized powder is sterile. Solvents may contain excipients that improve stability, or other pharmacological components of powders or reconstituted solutions prepared from powders. Excipients that can be used include, but are not limited to, dextrose, sorbitol, fructose, corn syrup, xylitol, glycerin, glucose, sucrose, or other suitable agents. The solvent may also contain, in one embodiment, a buffer of about neutral pH, such as citric acid, sodium or potassium phosphate, or other such buffer known to those of skill in the art. The desired formulation is provided by lyophilization following sterile filtration of the solution under standard conditions known to those of skill in the art. In one embodiment, the resulting solution is dispensed into lyophilized vials. Each vial will contain a single dose or multiple doses of the compound. The lyophilized powder can be stored under suitable conditions such as about 4 ° C. to room temperature. Reconstitution of this lyophilized powder with water for injection provides a formulation for use in parenteral administration. For reconstruction, the lyophilized powder is added to sterile water or other suitable carrier. The exact amount depends on the compound selected. Such an amount can be determined empirically.

The anti-ApoC3 antibodies disclosed herein and other compositions provided herein are also formulated to target specific tissues, receptors, or other parts of the body to be treated. Can be transformed into. Many such methods of targeting are well known to those of skill in the art. All such targeting methods are contemplated herein for use in this composition. For non-limiting examples of targeting methods, see, for example, US Pat. Nos. 6,316,652, 6,274,552, 6,271,359, 6,253,872. No. 6,139,865, No. 6,131,570, No. 6,120,751, No. 6,071,495, No. 6,060,082, No. 6 , 048,736, 6,039,975, 6,004,534, 5,985,307, 5,972,366, 5,900,252 , 5,840,674, 5,759,542, and 5,709,874.

The composition used for in vivo administration can be sterile. This is easily achieved, for example, by filtration through a sterile filter membrane.

5. Polynucleotides, Vectors, and Methods of Producing Anti-ApoC3 Antibodies In another embodiment, a polynucleotide comprising a nucleotide sequence encoding an anti-ApoC3 antibody disclosed herein (eg, a light chain variable region or a heavy chain variable region). , And vectors, eg, vectors containing such polynucleotides for recombinant expression in host cells (eg, E. coli and mammalian cells) are provided herein.

As used herein, an "isolated" polynucleotide or nucleic acid molecule is one separated from other nucleic acid molecules present in the natural source of the nucleic acid molecule (eg, in mice or humans). In addition, "isolated" nucleic acid molecules, such as cDNA molecules, are substantially free of culture medium or chemically synthesized when produced by other cellular trait components or recombinant techniques. In some cases, it may be substantially free of chemical precursors or other chemicals. For example, the expression "substantially free" is about 15%, 10%, 5%, 2%, 1%, 0.5%, or less than 0.1% (especially less than about 10%). Includes preparations of a substance, eg, a cellular material, a culture medium, another nucleic acid molecule, a chemical precursor, or a polynucleotide or nucleic acid molecule having another chemical. In certain embodiments, the nucleic acid molecule (s) encoding the antibodies disclosed herein are isolated or purified.

In a specific embodiment, an antibody that specifically binds to an ApoC3 (eg, human ApoC3) peptide and comprises an amino acid sequence disclosed herein, as well as such antibodies for binding to an ApoC3 (eg, human ApoC3) polypeptide. Provided herein are polypeptides comprising a nucleotide sequence encoding an antibody that competes with (eg, in a dose-dependent manner) or binds to the same epitope as the epitope of such antibody.

In certain embodiments, polynucleotides comprising a nucleotide sequence encoding a light chain or heavy chain of an antibody disclosed herein are provided herein. Polynucleotides may comprise a nucleotide sequence encoding the VH, VL, or CDR of an antibody disclosed herein (see, eg, Tables 1-4 herein).

Also provided herein are polynucleotides encoding anti-ApoC3 antibodies that are optimized, for example, by codon / RNA optimization, replacement with heterologous signal sequences, and elimination of mRNA instability elements. Optimized to encode anti-ApoC3 antibodies for recombinant expression (eg, light chain, heavy chain, VH domain, or VL domain) by introducing codon changes or eliminating inhibitory regions in mRNA. Methods for producing nucleic acids include, for example, US Pat. Nos. 5,965,726, 6,174,666, 6,291,664, 6,414,132, and 6. , 794, 498 can be carried out by appropriately adapting the optimization method described in No. 794, 498. For example, potential splice sites and unstable elements in RNA (eg, A / T or A / U rich elements) can be mutated without modification of the amino acid encoded by the nucleic acid sequence for recombinant expression. RNA stability can be increased. Modifications utilize the degeneracy of the genetic code, for example, using alternative codons of the same amino acid. In some embodiments, it may be desirable to modify one or more codons to encode a conservative mutation, eg, a similar amino acid having a chemical structure and properties or functions similar to the original amino acid. Such a method expresses anti-ApoC3 at least 1-fold, 2-fold, 4-fold, 5-fold, 10-fold, and 20-fold compared to the expression of anti-ApoC3 antibody encoded by a non-optimized polynucleotide. , 30 times, 40 times, 50 times, 60 times, 70 times, 80 times, 90 times, or 100 times or more.

In certain embodiments, the optimized polynucleotide sequence encoding the anti-ApoC3 antibody disclosed herein (eg, VL domain or VH domain) is the anti-ApoC3 antibody disclosed herein (eg, eg, VH domain). It is possible to hybridize to an anti-sense (eg, complement) polynucleotide of a non-optimized polynucleotide sequence encoding (VL domain or VH domain). In certain embodiments, the optimized nucleotide sequence encoding the anti-ApoC3 antibody or fragment disclosed herein is optimized to encode the anti-ApoC3 antibody disclosed herein under high stringency conditions. It hybridizes to the antisense polynucleotide of the unmodified polynucleotide sequence. In certain embodiments, the optimized nucleotide sequences encoding the anti-ApoC3 antibodies disclosed herein are disclosed herein under high stringency, medium or low stringency hybridization conditions. It hybridizes to an antisense polynucleotide with a non-optimized nucleotide sequence of anti-ApoC3 antibody. Information on hybridization conditions is described, see, for example, US Patent Application Publication No. 2005/0048549 (eg, paragraphs 72-73), which is incorporated herein by reference.

A polynucleotide can be obtained and the nucleotide sequence of the polynucleotide can be determined by any method known in the art. The antibodies disclosed herein, eg, the antibodies listed in Table 1, and the nucleotide sequences encoding modified versions of these antibodies, use methods well known in the art, ie, specific amino acids. Nucleotide codons known to encode an antibody can be aggregated and determined to produce a nucleic acid encoding an antibody. Such polynucleotides encoding antibodies can be assembled from chemically synthesized oligonucleotides (eg, as described in Kutmeier G et al., (1994), BioTechniques 17: 242-6). This simply involves the synthesis of overlapping oligonucleotides containing a portion of the sequence encoding the antibody, annealing and ligation of those oligonucleotides, and subsequent amplification by PCR of the ligated oligonucleotides.

Alternatively, the polynucleotide encoding the antibody disclosed herein is a suitable source (eg, PCR and other molecular cloning methods) using methods well known in the art (eg, PCR and other molecular cloning methods). It can be produced from nucleic acids from hybridomas). For example, genomic DNA obtained from hybridoma cells producing the antibody of interest can be used to perform PCR amplification using synthetic primers capable of hybridizing to the 3'and 5'ends of known sequences. can. Such PCR amplification methods can be used to obtain nucleic acids containing sequences encoding the light or heavy chains of an antibody. Such a PCR amplification method can be used to obtain a nucleic acid containing a sequence encoding a variable light chain region or a variable heavy chain region of an antibody. The amplified nucleic acid can be cloned into a vector for expression in a host cell and, for example, for further cloning to generate chimeric and humanized antibodies.

If a clone containing the nucleic acid encoding a particular antibody is not available, but the sequence of the antibody molecule is known, the nucleic acid encoding the immunoglobulin can be synthesized to hybridize to the 3'and 5'ends of the sequence. Suitable sources (eg, by PCR amplification using primers, or, for example, by cloning using a specific gene sequence-specific oligonucleotide probe to identify a cDNA clone from a cDNA library encoding an antibody. An antibody cDNA library generated from any tissue or cell expressing an antibody, such as an antibody cDNA library, or a hybridoma cell selected to express an antibody disclosed herein, or a nucleic acid isolated from it. , Preferably poly A + RNA), or can be obtained from it. The amplified nucleic acid produced by PCR can then be cloned into a replicable cloning vector using any method well known in the art.

The DNA encoding the anti-ApoC3 (eg, human ApoC3) antibody disclosed herein encodes the heavy and light chains of the anti-ApoC3 (eg, human ApoC3) antibody using conventional procedures (eg, human ApoC3). It can be easily isolated and sequenced (by using an oligonucleotide probe that can specifically bind to the gene). Hybridoma cells can serve as a source of such DNA. Once isolated, the DNA is placed in an expression vector, which in turn does not otherwise produce immunoglobulin proteins, E. coli. Transfected and recombinant into host cells such as colli cells, monkey COS cells, Chinese hamster ovary (CHO) cells (eg, CHO cells from CHO GS System® (Lonza)), or myeloma cells. Obtain synthesis of anti-ApoC3 (eg, human ApoC3) antibodies in host cells.

PCR primers containing VH or VL nucleotide sequences, restriction sites, and flanking sequences that protect the restriction sites can be used to amplify the VH or VL sequences of scFv clones to generate complete antibodies. Using cloning techniques known to those of skill in the art, PCR amplified VH domains can be cloned into vectors expressing heavy chain constant regions, eg, human gamma 4 constant regions, and PCR amplified VL domains can be obtained by light chain determination. It can be cloned into a vector expressing a normal region, eg, a human kappa or lambda constant region. In certain embodiments, the vector for expressing the VH or VL domain comprises a selectable marker such as an EF-1α promoter, a secretory signal, a cloning site for a variable region, a constant region, and neomycin. The VH and VL domains may also be cloned into a single vector expressing the required constant regions. The heavy and light chain conversion vectors are then co-transfected into the cell line using techniques known to those of skill in the art to produce stable or transient cell lines expressing full-length antibodies such as IgG. Generate.

DNA also covalently binds all or part of the coding sequence of a non-immunoglobulin polypeptide to, for example, by substituting the coding sequences of human heavy and light chain constant domains for mouse sequences, or to immunoglobulin coding sequences. Can be modified by letting.

Polynucleotides that hybridize to the polynucleotides encoding the antibodies disclosed herein under high, medium, or low stringency hybridization conditions are also provided. In certain embodiments, the polynucleotides disclosed herein are those encoding the VH or VL domains provided herein under high, medium, or low stringency hybridization conditions. Hybridize.

Hybridization conditions have been described in the art and are known to those of skill in the art. For example, hybridization under stringent conditions is hybridization to the filter-bound DNA in 6 × sodium chloride / sodium citrate (SSC) at about 45 ° C, followed by 0.2 × SSC at about 50-65 ° C. / May be accompanied by one or more washes in 0.1% SDS. Hybridization under high stringent conditions is hybridization to the filter-bound nucleic acid in 6 × SSC at about 45 ° C, followed by 1 in 0.1 × SSC / 0.2% SDS at about 68 ° C. May involve more than one wash. Hybridization under other stringent hybridization conditions is known and described by those of skill in the art, eg, Ausubel FM et al. , Eds. , (1989) Current Protocols in Molecular Biology, Vol. I, Green Publishing Associates, Inc. And John Wiley & Sons, Inc. , New York, pages 6.3.1-6.3.6 and 2.10.3.

In certain embodiments, cells (eg, host cells) expressing (eg, recombinantly) the antibodies disclosed herein that specifically bind to ApoC3 (eg, human ApoC3), as well as associated polynucleotides and Expression vectors are provided herein. Vectors (eg, expression vectors) comprising a polynucleotide comprising a nucleotide sequence encoding an anti-ApoC3 (eg, human ApoC3) antibody or fragment for recombinant expression in a host cell, preferably a mammalian cell, are described herein. Provided. Also provided are host cells comprising such a vector for recombinant expression of an ApoC3 (eg, human ApoC3) antibody (eg, a human or humanized antibody) disclosed herein. In certain embodiments, methods for producing the antibodies disclosed herein are provided herein, comprising expressing such antibodies from a host cell.

A set of antibodies disclosed herein (eg, full-length antibodies, heavy or light chains of antibodies, or single-chain antibodies disclosed herein) that specifically bind to ApoC3 (eg, human ApoC3). Replacement expression involves the construction of an expression vector containing a polynucleotide encoding an antibody. Once a polynucleotide encoding an antibody molecule, heavy chain or light chain (eg, heavy chain or light chain variable region) of an antibody disclosed herein is obtained, the vector for the production of the antibody molecule is such technique. It can be produced by recombinant DNA technology using techniques well known in the art. Accordingly, methods for preparing a protein by expressing a polynucleotide containing an antibody or antibody fragment (eg, light chain or heavy chain) encoding a nucleotide sequence are disclosed herein. Methods well known to those of skill in the art can be used to construct expression vectors containing antibodies or antibody fragments (eg, light or heavy chains) that encode sequences as well as appropriate transcriptional and translational control signals. These methods include, for example, in vitro recombinant DNA techniques, synthetic techniques, and in vivo gene recombination. Also, an antibody molecule disclosed herein operably linked to a promoter, a heavy or light chain of an antibody, a heavy or light chain variable region of an antibody, or a nucleotide sequence encoding a heavy or light chain CDR. A replicable vector containing the above is also provided. Such vectors may include, for example, a nucleotide sequence encoding a constant region of an antibody molecule (see, eg, WO 86/05807 and WO 89/01036, and US Pat. No. 5,122,464). Variable regions of the antibody may be included and cloned into such vectors for expression of whole heavy chains, whole light chains, or both heavy chains and whole light chains.

The expression vector can be transferred to a cell (eg, a host cell) by conventional techniques and the resulting cells can then be cultured by conventional techniques to produce the antibodies disclosed herein. Accordingly, an antibody disclosed herein, or a heavy or light chain thereof, or a fragment thereof, or a fragment thereof, disclosed herein operably linked to a promoter for expression of such a sequence in a host cell. Host cells containing polynucleotides encoding single chain antibodies are provided herein. In certain embodiments, in the case of double-stranded antibody expression, the vectors encoding both heavy and light chains are individually hosts for expression of the entire immunoglobulin molecule, as detailed below. Can be co-expressed in cells. In certain embodiments, the host cell comprises a vector comprising a polynucleotide encoding both the heavy and light chains of the antibody disclosed herein. In certain embodiments, the host cell comprises a first vector comprising a polynucleotide encoding a heavy chain or heavy chain variable region of an antibody or fragment thereof disclosed herein, and an antibody disclosed herein. Or contains two different vectors, a second vector comprising a polynucleotide encoding a light chain or light chain variable region of the fragment. In another embodiment, the first host cell comprises a first vector comprising a heavy chain or a heavy chain variable region encoding a heavy chain or heavy chain variable region of an antibody or fragment thereof disclosed herein, the second host. The cell comprises a second vector containing a polynucleotide encoding a light chain or light chain variable region of an antibody disclosed herein. In certain embodiments, the heavy chain / heavy chain variable region expressed by the first cell associates with the light chain / light chain variable region of the second cell to the anti-ApoC3 antibody disclosed herein. Formed. In certain embodiments, a population of host cells comprising such a first host cell and such a second host cell is provided herein.

In certain embodiments, a first vector comprising a polynucleotide encoding a light chain / light chain variable region of an anti-ApoC3 antibody disclosed herein and a heavy chain of the anti-ApoC3 antibody disclosed herein. A second vector comprising a polynucleotide encoding a heavy chain variable region and a population of vectors comprising the / heavy chain variable region are provided herein.

A variety of host expression vector systems can be used to express the antibody molecules disclosed herein (see, eg, US Pat. No. 5,807,715). Such a host expression system represents a vehicle in which the coding sequence of interest is produced and can be subsequently purified, but in situ the antibody molecules disclosed herein when transformed or transfected with the appropriate nucleotide coding sequence. Also represents cells that can be expressed in. These include microorganisms such as recombinant bacteriophage DNA, plasmid DNA, or bacteria transformed with a cosmid DNA expression vector (eg, E. coli and B. subtilis) containing antibody coding sequences; antibody coding sequences. Contains recombinant yeast expression vector-transformed yeast (eg, Saccharomyces Pichia); recombinant virus expression vector containing recombinant virus expression vector (eg, baculovirus) -infected insect cell line; antibody coding sequence. A plant cell line containing a recombinant virus expression vector (eg, Califlower Mosaic Virus, CaMV; Tobacco Mosaic Virus, TMV) or transformed with a recombinant plasmid expression vector (eg, Ti plasmid) (eg, Ti plasmamid). , A green alga such as a plasmid (plasmidomonas reinhardtii); Mammalian cell lines carrying recombinant expression constructs (eg, COS (eg, COS1 or COS), CHO, BHK, MDCK, HEK 293, NS0, PER.C6, VERO, CRL7030, HsS78Bst, HeLa, and NIH 3T3, Includes, but is not limited to, HEK-293T, HepG2, SP210, R1.1, BW, LM, BSC1, BSC40, YB / 20, and BMT10 cells). In certain embodiments, the cells for expressing the antibodies disclosed herein are CHO cells, eg, CHO cells from CHO GS System® (Lonza). In certain embodiments, the cell for expressing the antibodies disclosed herein is a human cell, eg, a human cell line. In certain embodiments, the mammalian expression vector is pOptiVEC® or pcDNA3.3. In certain embodiments, bacterial cells such as Escherichia coli or eukaryotic cells (eg, mammalian cells) are used for the expression of recombinant antibody molecules, especially in the case of expression of whole recombinant antibody molecules. For example, mammalian cells such as Chinese hamster ovary (CHO) cells are an effective expression system for antibodies, along with vectors such as the early gene promoter element, which is a major intermediate from human cytomegalovirus. Fooking MK & Hofstetter H (1986) Gene 45: 101-5 and Cockett MI et al., (1990) Biotechnology 8 (7): 662-7). In certain embodiments, the antibodies disclosed herein are produced by CHO cells or NS0 cells. In certain embodiments, expression of a nucleotide sequence encoding an antibody disclosed herein that specifically binds to ApoC3 (eg, human ApoC3) is carried out by a constitutive promoter, an inducible promoter, or a tissue-specific promoter. Be controlled.

In a bacterial system, several expression vectors may be advantageously selected, depending on the intended use for the expressed antibody molecule. For example, if large quantities of such antibodies are produced, vectors that direct the expression of readily purified high levels of fusion protein products may be desirable for the production of pharmaceutical compositions of antibody molecules. Such a vector can be individually ligated into the vector in a frame having a lac Z coding region such that the fusion protein is produced. coli expression vector pUR278 (Ruether U & Muller-Hill B (1983) EMBO J 2: 1791-1794); pIN vector (Inouye S & Inouye M (1985) Nuc Acids Res 13: 3101-3109, Van Heke 24: 5503-5509) and the like, but are not limited thereto. For example, the pGEX vector can also be used to express a foreign polypeptide as a fusion protein with glutathione 5-transferase (GST). In general, such fusion proteins are soluble and can be easily purified from lysed cells by elution in the presence of free glutathione following adsorption and binding to matrix glutathione agarose beads. Since the pGEX vector is designed to contain a thrombin or factor Xa protease cleavage site, the cloned target gene product is released from the GST moiety.

In an insect system, for example, Autographa californica nuclear polyhedrosis virus (AcNPV) can be used as a vector for expressing a foreign gene. The virus propagates in Prodenia flugiperda cells. The antibody coding sequence can be individually cloned into a non-essential region of the virus (eg, the polyhedrin gene) and placed under the control of the AcNPV promoter (eg, the polyhedrin promoter).

Several virus-based expression systems are available in mammalian host cells. When adenovirus is used as the expression vector, the antibody coding sequence of interest can be ligated to an adenovirus transcription / translation control complex, eg, a late promoter and a tripartite leader sequence. The chimeric gene can then be inserted into the adenovirus genome by in vitro or in vivo recombination. Insertion into a non-essential region of the viral genome (eg, region E1 or E3) will result in a recombinant virus that is viable in the infected host and capable of expressing antibody molecules (eg, Logan). See J & Schenk T (1984) PNAS 81 (12): 3655-9). Also, a specific initiation signal may be required for efficient translation of the inserted antibody coding sequence. These signals include the ATG start codon and adjacent sequences. In addition, the start codon must coincide with the reading frame of the desired coding sequence to ensure translation of the entire insert. These extrinsic translational control signals and start codons can be of various origins, both natural and synthetic. The effectiveness of expression can be enhanced by including appropriate transcription enhancer elements, transcription terminators, etc. (see, eg, Bitter G et al., (1987) Methods Enzymol. 153: 516-544).

In addition, host cell lines can be selected that regulate the expression of the inserted sequence or modify and process the gene product in a particular manner desired. Such modifications (eg, glycosylation) and processing (eg, cleavage) of the protein product can be important for the function of the protein. Different host cells have characteristic and specific mechanisms for post-translational processing and modification of proteins and gene products. Appropriate cell lines or host systems can be selected to ensure correct modification and processing of expressed foreign proteins. For this purpose, eukaryotic host cells can be used that possess the cellular mechanisms for proper processing, glycosylation, and phosphorylation of the primary transcript of the gene product. Such mammalian host cells include CHO, VERO, BHK, Hela, MDCK, HEK293, NIH3T3, W138, BT483, Hs578T, HTB2, BT2O and T47D, NS0 (mouse that endogenously does not produce any immunoglobulin chain). Myeloma cell line), CRL7O3O, COS (eg, COS1 or COS), PER. Includes, but is not limited to, C6, VERO, HsS78Bst, HEK-293T, HepG2, SP210, R1.1, BW, LM, BSC1, BSC40, YB / 20, BMT10, and HsS78Bst cells. In certain embodiments, the anti-ApoC3 (eg, human ApoC3) antibodies disclosed herein are produced in mammalian cells such as CHO cells.

In certain embodiments, the antibodies disclosed herein have a reduced fucose content or do not contain fucose. Such antibodies can be produced using techniques known to those of skill in the art. For example, the antibody can be expressed in cells that lack or lack the ability to fucosylate. In certain examples, cell lines with knockouts of both α1,6-fucosyltransferase alleles can be used to produce antibodies with reduced fucose content. The Potelligent® system (Lonza) is an example of a system that can be used to produce antibodies with reduced fucose content.

Stable expression cells can be produced for long-term, high-yield production of recombinant proteins. For example, cell lines that stably express the anti-ApoC3 antibody disclosed herein can be engineered. In certain embodiments, the cells provided herein are stable in a light chain / light chain variable region and a heavy chain / heavy chain variable region that are associated to form the antibodies disclosed herein. Express.

In certain embodiments, instead of using an expression vector containing a viral replication origin, the host cell can be selected with appropriate expression control elements (eg, promoters, enhancers, sequences, transcription terminators, polyadenylation sites, etc.). It can be transformed with DNA controlled by a promoter. After introduction of the foreign DNA / polynucleotide, the modified cells are grown in concentrated medium for 1-2 days and then switched to selective medium. Selectable markers for recombinant plasmids confer resistance to selection, allowing cells to stably integrate the plasmid into the chromosome, proliferate, and eventually form a focus that can be cloned into a cell line for proliferation. enable. This method can be advantageously used to engineer cell lines expressing the anti-ApoC3 antibodies disclosed herein. Such engineered cell lines may be particularly useful in screening and evaluation of compositions that interact directly or indirectly with antibody molecules.

Several selection systems can be used, which include simple herpesvirus thymidine kinase (Willer M et al., (1977) Cell 11 (1): in tk cells, hgprt cells, or aprt cells, respectively. 223-32), hypoxanthine annin phosphoribosyl transferase (Szybalska EH & Szybalski W (1962) PNAS 48 (12): 2026-2034), and adenine phosphoribosyl transferase (Lowy I et al., (1980) Cell 22 (3) :. 817-23) Genes are included, but not limited to these. Metabolism antagonist resistance can also be used as the basis for selection for the following genes: dhfr (Wiggler M et al., (1980) PNAS 77 (6): 3567-70, O'Hare K), which confer resistance to methotrexate. et al., (1981) PNAS 78: 1527-31); resistance to mycophenolic acid gpt (Mulligan RC & Berg P (1981) PNAS 78 (4): 2072-6); resistance to aminoglycosides G-418. Neo (Wu GY & Wu CH (1991) Biotherapy 3: 87-95, Tolstoshev P (1993) Ann Rev Pharmacol Toxicol 32: 573-596, Mulligan RC (1993) Science 260: 1993) Ann Rev Biochem 62: 191-217, Nabel GJ & Fergner PL (1993) Trends Biotechnol 11 (5): 211-5); and hygro (Santerre RF et al., (1984) gen. (1-3): 147-56). Methods generally known in the art of recombinant DNA technology can be routinely applied to select the desired recombinant clone, and such methods are described, for example, in Ausubel FM et al. , (Eds.), Current Protocols in Molecular Biology, John Wiley & Sons, NY (1993), Kriegler M, Gene Transfer and Expression, Et al. .. , (Eds.), Current Protocols in Human Genetics, John Wiley & Sons, NY (1994), Colbere-Garapin F et al. , (1981) J Mol Biol 150: 1-14, which are incorporated herein by reference in their entirety.

The expression level of the antibody molecule can be increased by vector amplification (for overview, Bebbington CR & Hentschel CCG, The if vector's based on gene amplification for the expression, cloning of cloning, cloning, cloning, cloning, and cloning. , New York, 1987)). When the vector-based marker expressing the antibody is amplifyable, increasing the level of inhibitor present in the culture of the host cell will increase the number of copies of the marker gene. Since the amplified region is associated with the antibody gene, antibody production will also be increased (Crowse GF et al., (1983) Mol Cell Biol 3: 257-66).

Host cells are simultaneously transfected with two or more expression vectors described herein, a first vector encoding a heavy chain-derived polypeptide and a second vector encoding a light chain-derived polypeptide. can do. The two vectors may contain the same selectable markers, which allow for equivalent expression of heavy and light chain polypeptides. Host cells can be simultaneously transfected with different amounts of two or more expression vectors. For example, in the host cell, the ratio of the first expression vector to the second expression vector is 1: 1, 1: 2, 1: 3, 1: 4, 1: 5, 1: 6, 1: 7, 1: 1. 8, 1: 9, 1:10, 1:12, 1:15, 1:20, 1:25, 1:30, 1:35, 1:40, 1:45, or 1:50 It can be transfected with one or the other.

Alternatively, a single vector can be used that encodes both heavy and light chain polypeptides and is capable of expressing them. In such situations, a light chain should be placed in front of the heavy chain to avoid heavy chains that do not have excessive toxicity (Proudfoot NJ (1986) Nature 322: 562-565 and Kohler G (1980) PNAS. 77: 2197-2199). The heavy and light chain coding sequences can include cDNA or genomic DNA. The expression vector can be monocistron or multicistron. The multicistron nucleic acid construct encodes a gene / nucleotide sequence in the range of 2, 3, 4, 5, 6, 7, 8, 9, 10 or more, or 2-5, 5-10, or 10-20. be able to. For example, bicistron nucleic acid constructs are disclosed in the following order: promoter, first gene (eg, heavy chain of antibody disclosed herein), and second gene and (eg, herein). The light chain of the antibody to be produced) may be included. In such expression vectors, transcription of both genes can be driven by a promoter, while translation of mRNA from the first gene can be due to a cap-dependent scanning mechanism, translation of mRNA from the second gene. , Cap-dependent mechanism, eg IRES.

Once the antibody molecule disclosed herein is produced by recombinant expression, it can be produced, for example, by any method known in the art for purification of immunoglobulin molecules, eg, chromatography (eg, ion). Purified by exchange, affinity, particularly affinity for a particular antigen after protein A, and size column chromatography), centrifugation, differential solubility, or any other standard technique for purification of the protein. obtain. In addition, the antibodies disclosed herein can be fused to a heterologous polypeptide sequence disclosed herein or otherwise known in the art to facilitate purification. ..

In certain embodiments, the antibodies disclosed herein have been isolated or purified. In general, an isolated antibody is substantially free of other antibodies having antigen specificity different from that of the isolated antibody. For example, in certain embodiments, the antibody preparations disclosed herein are substantially free of cellular material or chemical precursors. The expression "substantially free of cellular material" includes a preparation of the antibody from which the antibody has been isolated or recombinantly produced from the cellular components of the cell. Thus, antibodies that are substantially free of cellular material are about 30%, 20%, 10%, 5%, 2%, 1%, 0.5%, or less than 0.1% (by dry weight). Preparations of antibodies with heterologous proteins (also referred to herein as "mixed proteins"), or variants of antibodies, such as different post-translational modified forms of antibodies or other different versions of antibodies (eg, antibody fragments). Is included. When the antibody is recombinantly produced, it also generally contains substantially no culture medium, i.e., the culture medium is approximately 20%, 10%, 2%, 1%, 0% of the volume of the protein preparation. It accounts for 5% or less than 0.1%. When an antibody is produced by chemical synthesis, it generally contains virtually no chemical precursors or other chemicals, ie it is a chemical precursor or other chemical involved in the synthesis of the protein. Is separated from. Thus, such preparations for antibodies have about 30%, 20%, 10%, or less than 5% (by dry weight) chemical precursors or compounds other than the antibody of interest. In certain embodiments, the antibodies disclosed herein have been isolated or purified.

Antibodies that specifically bind to ApoC3 (eg, human ApoC3) can be produced by any method known in the art for the synthesis of the antibody, eg, by chemical synthesis, or by recombinant expression techniques. Unless otherwise indicated, the methods disclosed herein are molecular biology, microbiology, gene analysis, recombinant DNA, organic chemistry, biochemistry, PCR, oligonucleotide synthesis and modification, nucleic acid hybridization, and techniques thereof. Use conventional techniques in related fields within the field. These techniques are described, for example, in the references cited herein and are fully described in the literature. For example, Maniatis T et al. , (1982) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Sambrook J et al. , (1989), Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press, Sambrook Jet al. , (2001) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, Ausubel FM et al. , Current Protocols in Molecular Biology, John Wiley & Sons (1987 and annual updates), Current Protocols in Immunology, John Wiley & Sons (1987 and annual updates) Gait (1984) Oligonucleotide Synthesis (ed.): A Practical Approach, IRL Press, Eckstein (ed (1991) Olympicotides and Analogues: A Practical Approach, IRL Press, Birren B et al. , (Eds.) (1999) Genome Analysis: A Laboratory Manual, Cold Spring Harbor Laboratory Press.

In certain embodiments, the antibodies disclosed herein are antibodies prepared, expressed, prepared, or isolated by any means involving, for example, synthesis, genetic manipulation-mediated production of DNA sequences (eg,). Recombinant antibody). In certain embodiments, such antibodies include sequences (eg, DNA or amino acid sequences) that are not naturally present in the antibody germline repertoire of animals or mammals (eg, humans) in vivo.

In one aspect, a method of making an antibody that specifically binds to ApoC3 (eg, human ApoC3), comprising culturing the cells or host cells disclosed herein, is described herein. Provided. In certain embodiments, a method of making an antibody that specifically binds to ApoC3 (eg, human ApoC3), wherein the cell or host cell disclosed herein (eg, the antibody disclosed herein). Provided herein are methods comprising expressing an antibody (eg, recombinant expression) using a cell containing the encoding polynucleotide or a host cell). In certain embodiments, the cell is an isolated cell. In certain embodiments, exogenous polynucleotides have been introduced into the cell. In certain embodiments, the method further comprises the step of purifying an antibody obtained from a cell or host cell.

Methods for producing polyclonal antibodies are known in the art (eg, Short Protocols in Molecular Biology, (2002) 5th Ed., Ausubel FM et al., Eds., John Wiley and Sons, New York. See Chapter 11).

Monoclonal antibodies can be prepared using a wide range of techniques known in the art, including the use of hybridoma, recombination, and phage display techniques, or combinations thereof. For example, monoclonal antibodies are known in the art and are described, for example, in Harlow E & Lane D, Antibodies: A Laboratory Manual, (Cold Spring Harbor Laboratory Press, 2nd ed. 1988), Hamm. , In: Monoclonal Antibodies and T-Cell Hybridomas 563 681 (Elsevier, NY, 1981) can be produced using hybridoma techniques, including those taught. The term "monoclonal antibody" as used herein is not limited to antibodies produced by hybridoma technology. For example, a monoclonal antibody can be recombinantly produced from an antibody disclosed herein, eg, a host cell that exogenously expresses the light or heavy chain of such antibody.

In certain embodiments, as used herein, a "monoclonal antibody" is produced by a single cell (eg, a hybridoma or host cell that produces a recombinant antibody), and the antibody is, for example, an ELISA. Alternatively, it specifically binds to ApoC3 (eg, human ApoC3) as is known in the art or as determined by other antigen binding or competitive binding assays in the examples provided herein. In certain embodiments, the monoclonal antibody can be a chimeric antibody or a humanized antibody. In certain embodiments, the monoclonal antibody is a monovalent antibody or a multivalent (eg, divalent) antibody. In certain embodiments, the monoclonal antibody is a monospecific or multispecific antibody (eg, a bispecific antibody). Monoclonal antibodies disclosed herein can be made, for example, by the hybridoma method described in Kohler G & Milstein C (1975) Nature 256: 495, or, for example, phage using the techniques disclosed herein. Can be isolated from the library. Clone cell lines and other methods for the preparation of monoclonal antibodies expressed thereby are well known in the art (eg, Short Protocols in Molecular Biology, (2002) 5th Ed., Ausubel FM et al. , (See Chapter 11 above)).

Methods for producing and screening specific antibodies using hybridoma technology are conventional and well known in the art. For example, in a hybridoma method, a protein used for immunization by immunizing a mouse or other suitable host animal such as a sheep, goat, rabbit, rat, hamster, or mackerel monkey (eg, ApoC3 (eg, human)). It produces or induces lymphocytes capable of producing antibodies that will specifically bind to ApoC3)). Alternatively, lymphocytes can be immunized in vitro. Then, lymphocytes are fused to myeloma cells using a suitable fusion agent such as polyethylene glycol to form hybridoma cells (Goding JW (Ed), Monoclonal Antibodies: Principles and Practice, pp. 59-103 (). Academic Press, 1986)). In addition, RIMMS (Repeated Immunization Multiple Sites) technology can be used to immunize animals (Kilpatrick KE et al., (1997) Hybridoma 16: 381-9, which is incorporated by reference in its entirety).

In some embodiments, mice (or other animals such as rats, monkeys, donkeys, pigs, sheep, hamsters, or dogs) can be immunized with an antigen (eg, ApoC3 (eg, human ApoC3)). Once an immune response was detected (eg, antigen-specific antibodies were detected in mouse serum), mouse spleens were harvested and splenocytes were isolated. The splenocytes are then fused by well known techniques to any suitable myeloma cell, eg, the cell line SP20 available from the American Type Culture Collection (ATCC®) (Manassas, VA) to form a hybridoma. do. Hybridomas are selected and cloned by limiting dilution. In certain embodiments, lymph nodes of immunized mice are harvested and fused with NS0 myeloma cells.

Hybridoma cells thus prepared are preferably seeded and grown in a suitable culture medium containing one or more substances that inhibit the growth or survival of unfused myeloma cells. For example, if the parent myeloma cells lack the enzyme hypoxanthine anine phosphoribosyl transferase (HGPRT or HPRT), the culture medium for hybridomas will typically contain hypoxanthine, aminopterin, and thymidine ( HAT medium), these substances prevent the growth of HGPRT-deficient cells.

Certain embodiments use myeloma cells that are efficiently fused, support stable, high levels of antibody production by selected antibody-producing cells, and are sensitive to media such as HAT medium. Among these myeloma cell lines are mouse myeloma cell lines, such as NS0 cell lines, or MOPC-21 and MPC-11 mouse tumors available from the Sark Institute Cell Distribution Center (San Diego, CA, USA). , And those derived from SP-2 or X63-Ag8.653 cells available from American Type Culture Collection (Rockville, MD, USA). Human myeloma and mouse-human heteromyeloma cell lines have also been described for the production of human monoclonal antibodies (Kozbor D (1984) J Immunol 133: 3001-5, Brodeur et al., Monoclonal Antibodies Production Technology, Technology and Analysis. pp. 51-63 (Marcel Decker, Inc., New York, 1987).

Culture medium growing hybridoma cells is assayed for the production of monoclonal antibodies against ApoC3 (eg, human ApoC3). The binding specificity of monoclonal antibodies produced by hybridoma cells can be determined by methods known in the art, such as by immunoprecipitation or in vitro such as radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA). Determined by binding assay.

After identification of hybridoma cells that produce antibodies of the desired specificity, affinity, or activity, the clones can be subcloned by limiting dilution procedures and propagated by standard methods (Goding JW (Ed), Monoclonal Antibodies: Principles and Practices). (the above)). Suitable culture media for this purpose include, for example, D-MEM or RPMI 1640 medium. In addition, hybridoma cells can grow in vivo as ascites tumors in animals.

Monoclonal antibodies secreted by subclones are preferably cultured media by conventional immunoglobulin purification procedures such as, for example, protein A-sepharose, hydroxylapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography. , Stomach fluid, or serum.

Antibodies disclosed herein include antibody fragments that recognize specific ApoC3 (eg, human ApoC3) and can be produced by any technique known to those of skill in the art. For example, the Fab and F (ab') ₂ fragments disclosed herein use enzymes such as papain (to produce the Fab fragment) or pepsin (to produce the F (ab') ₂ fragment). It can be produced by proteolytic cleavage of immunoglobulin molecules. The Fab fragment corresponds to one of two identical arms of the antibody molecule and contains a complete light chain paired with the heavy VH and CH1 domains. The F (ab') ₂ fragment contains two antigen-binding arms of an antibody molecule bound by a disulfide bond in the hinge region.

In addition, the antibodies disclosed herein can also be produced using various phage display methods known in the art. In the phage display method, functional antibody domains are presented on the surface of phage particles carrying the polynucleotide sequences encoding them. In particular, the DNA sequences encoding the VH and VL domains are amplified from animal cDNA libraries (eg, human or mouse cDNA libraries of affected tissue). The DNA encoding the VH and VL domains is recombined with the scFv linker by PCR and cloned into a phagemid vector. Vector to E. Electroporate to colli and E. Infect colli with helper phage. The phage used in these methods are typically fibrous phage containing fd and M13, and the VH and VL domains are usually recombinantly fused to either the phage gene III or the gene VIII. Phage expressing an antigen-binding domain that binds to a particular antigen can be selected or identified using an antigen, eg, a labeled antigen, or an antigen bound or captured on a solid surface or beads. Examples of phage display methods that can be used to generate the antibodies disclosed herein include Brinkman U et al. , (1995) J Immunol Methods 182: 41-50, Ames RS et al. , (1995) J Immunol Methods 184: 177-186, Kettlebough CA et al. , (1994) Eur J Immunol 24: 952-958, Persic L et al. , (1997) Gene 187: 9-18, Burton DR & Barbas CF (1994) Advan Immunol 57: 191-280, PCT Application No. PCT / GB91 / 00134, International Publication No. WO90 / 02809, WO91 / 10737. No., WO92 / 01047, WO92 / 18619, WO93 / 11236, WO95 / 15982, WO95 / 20401, and WO97 / 13844, and US Patent No. 5. , 698,426, 5,223,409, 5,403,484, 5,580,717, 5,427,908, 5,750,753 , No. 5,821,047, No. 5,571,698, No. 5,427,908, No. 5,516,637, No. 5,780,225, No. 5, 658,727, 5,733,743, and 5,969,108 are disclosed.

As described in the references above, after phage selection, the antibody coding region from the phage is isolated and used to generate all antibodies, including human antibodies, or any other desired antigen-binding fragment. And can be expressed in any desired host, including, for example, mammalian cells, insect cells, plant cells, yeasts, and bacteria, as described below. PCT Publication No. WO 92/22324, Mullinax RL et al. , (1992) BioTechniques 12 (6): 864-9, Sawai Het al. , (1995) Am J Reprod Immunol 34: 26-34, and Better M et al. , (1988) Recombinant production of antibody fragments such as Fab, Fab', and F (ab') ₂ fragments using methods known in the art, such as those disclosed in Science 240: 1041-1403. Technology can also be used.

In certain embodiments, VH or VL sequences from templates such as scFv clones are used to generate complete antibodies using PCR primers containing VH or VL nucleotide sequences, restriction sites, and flanking sequences that protect the restriction sites. Can be amplified. Using cloning techniques known to those of skill in the art, PCR amplified VH domains can be cloned into vectors expressing VH constant regions, and PCR amplified VL domains can be expressed in VL constant regions such as human kappa or lambda constants. It can be cloned into a vector that expresses the region. The VH and VL domains can also be cloned into a single vector that expresses the required constant regions. Stable or transient cell lines that are then co-transfected into cell lines using techniques known to those of skill in the art to express full-length antibodies such as IgG. To generate.

Chimeric antibodies are molecules in which different parts of the antibody are derived from different immunoglobulin molecules. For example, a chimeric antibody may contain a variable region of a mouse or rat monoclonal antibody fused to the constant region of a human antibody. Methods for producing chimeric antibodies are known in the art. For example, Morrison SL (1985) Science 229: 1202-7, Oi VT & Morrison SL (1986) BioTechniques 4: 214-221, Gillies SD et al. , (1989) J Immunol Methods 125: 191-202, and US Pat. Nos. 5,807,715, 4,816,567, 4,816,397, and 6,331,415. Please refer to the issue.

The humanized antibody can bind to a given antigen and has substantially a framework region having the amino acid sequence of human immunoglobulin and substantially the amino acid sequence of non-human immunoglobulin (eg, mouse immunoglobulin). Includes CDR. In certain embodiments, the humanized antibody also comprises at least a portion of an immunoglobulin constant region (Fc) (typically that of a human immunoglobulin). The antibody can also include the CH1, hinge, CH2, CH3, and CH4 regions of the heavy chain. Humanized antibodies can be selected from any class of immunoglobulins, including IgM, IgG, IgD, IgA, and IgE, as well as any isotype, including IgG ₁ , IgG ₂ , IgG ₃ , and IgG ₄ . Humanized antibodies include CDR transplants (European Patent EP239400, International Publication No. WO91 / 09967, and US Pat. Nos. 5,225,539, 5,530,101, and 5,585,089. ), Veneering or resurfacing (European Pat. Nos. EP592106 and EP519596, Padlan EA (1991) Mol Immunol 28 (4/5): 489-498, Studnikka GM et al., (1994) Prot Engineering 7 (6): 805-814, and Roguska MA et al., (1994) PNAS 91: 969-973), Chain Shuffling (US Pat. No. 5,565,332), and, for example, the United States. Patent No. 6,407,213, US Pat. No. 5,766,886, International Publication No. WO93 / 17105, Tan Pet al. , (2002) J Immunol 169: 1119-25, Caldas C et al. , (2000) Protein Eng. 13 (5): 353-60, Morea V et al. , (2000) Methods 20 (3): 267-79, Baca M et al. , (1997) J Biol Chem 272 (16): 10678-84, Roguska MA et al. , (1996) Protein Eng 9 (10): 895 904, Couto JR et al. , (1995) Cancer Res. 55 (23 Supp): 5973s-5977s, Couto JR et al. , (1995) Cancer Res 55 (8): 1717-22, Sandhu JS (1994) Gene 150 (2): 409-10, and Pedersen JT et al. , (1994) J Mol Biol 235 (3): 959-73, but not limited to these, can be produced using a variety of techniques known in the art. See also U.S. Patent Application Publication No. 2005/0042664A1 (February 24, 2005), which is incorporated herein by reference in its entirety.

Methods for making multispecific (eg, bispecific antibodies) have been described, eg, US Pat. Nos. 7,951,917, 7,183,076, 8,227. , 577, 5,837,242, 5,989,830, 5,869,620, 6,132,992, and 8,586,713. Please refer.

Single domain antibodies, such as antibodies lacking a light chain, can be produced by methods well known in the art. Richmann L & Myldermans S (1999) J Immunol 231: 25-38, Nuttall SD et al. , (2000) Curr Pharma Biotechnol 1 (3): 253-263, Muyldermans S, (2001) J Biotechnol 74 (4): 277-302, US Pat. No. 6,005,079, and WO94 / 04678. No., WO94 / 25591, and WO01 / 44301.

In addition, antibodies that specifically bind to ApoC3 (eg, human ApoC3) can also be utilized to generate anti-idiotype antibodies that "mimic" the antigen, using techniques well known to those of skill in the art. Can be done. (See, for example, Greenspan NS & Bona CA (1989) FASEB J 7 (5): 437-444 and Nissinoff A (1991) J Immunol 147 (8): 2429-2438).

In certain embodiments, the antibody disclosed herein is a human antibody that binds to the same epitope of ApoC3 (eg, human ApoC3) as the anti-ApoC3 antibody disclosed herein. In certain embodiments, it is disclosed herein that competitively block binding (eg, in a dose-dependent manner) to ApoC3 (eg, human ApoC3) of any one of the antibodies disclosed herein. The antibody is a human antibody. Human antibodies can be produced using any method known to those of skill in the art. For example, transgenic mice that are unable to express functional endogenous immunoglobulins but are capable of expressing human immunoglobulin genes can be used. In particular, human heavy and light chain immunoglobulin gene complexes can be introduced into mouse embryonic stem cells at random or by homologous recombination. Alternatively, in addition to human heavy and light chain genes, human variable, constant, and diverse regions can be introduced into mouse embryonic stem cells. Mouse heavy and light chain immunoglobulin genes may become non-functional separately or simultaneously with the introduction of the human immunoglobulin locus by homologous recombination. In particular, homozygous deletions in the _JH region prevent endogenous antibody production. Modified embryonic stem cells are proliferated and microinjected into blastocysts to produce chimeric mice. The chimeric mice are then bred to produce homozygous offspring expressing human antibodies. Transgenic mice are immunized in the usual manner with all or part of the selected antigen, eg, an antigen (eg, ApoC3 (eg, human ApoC3)). Monoclonal antibodies to the antigen can be obtained from immunized transgenic mice using conventional hybridoma techniques. Human immunoglobulin transgenes carried by transgenic mice are reconstituted during B cell differentiation and subsequently undergo class switching and somatic mutations. Thus, such techniques can be used to produce therapeutically useful IgG, IgA, IgM, and IgE antibodies. For an overview of this technique for producing human antibodies, see Lomberg N & Huszar D (1995) Int Rev Immunol 13: 65-93. For a detailed discussion of this technique for producing human and human monoclonal antibodies, as well as the protocol for producing such antibodies, see, for example, WO 98/24893, WO 96/34096, and WO 96/34096. WO96 / 33735, and US Pat. Nos. 5,413,923, 5,625,126, 5,633,425, 5,569,825, 5,661,016. No. 5,545,806, No. 5,814,318, and No. 5,939,598. Examples of mice capable of producing human antibodies include Xenomouse® (Abgenix, Inc., US Pat. Nos. 6,075,181 and 6,150,184), HuAb-Mouse (Registered). Trademarks) (Mederex, Inc./GenPharm, US Pat. Nos. 5,545,806 and 5,569,825), Trans Chromo Mouse® (Kirin), and KM Mouse® (Registered Trademarks). Medarex Kirin).

Human antibodies that specifically bind to ApoC3 (eg, human ApoC3) can be made by a variety of methods known in the art, including the phage display method described above using antibody libraries derived from human immunoglobulin sequences. .. U.S. Pat. Nos. 4,444,887, 4,716,111, and 5,885,793, as well as International Publications WO98 / 46645, WO98 / 50433, and WO98 / 24893. See also WO98 / 16654, WO96 / 34096, WO96 / 33735, and WO91 / 10741.

In some embodiments, human antibodies can be produced using mouse-human hybridomas. For example, human peripheral blood lymphocytes transformed with Epstein-Barvirus (EBV) can be fused with mouse myeloma cells to produce mouse-human hybridomas that secrete human monoclonal antibodies, these mice-. Human hybridomas can be screened to determine which secretes a human monoclonal antibody that specifically binds to a target antigen (eg, ApoC3 (eg, human ApoC3)). Such methods are known and have been described in the art (eg, Shinmoto H et al., (2004) Cytotechnology 46: 19-23, Naganawa Y et al., (2005) Human Antibodies 14: 27-31). Please refer to).

6. Kits Kits are also provided that include one or more antibodies or pharmaceutical compositions or conjugates thereof disclosed herein. In certain embodiments, a pharmaceutical pack comprising one or more containers filled with one or more components of the pharmaceutical composition disclosed herein, such as one or more antibodies provided herein. Alternatively, the kit is provided herein. In some embodiments, the kit comprises a pharmaceutical composition disclosed herein and any prophylactic or therapeutic agent, such as those disclosed herein. Accompanying such containers as needed may be a notice in the form prescribed by a government agency that regulates the manufacture, use, or sale of medicinal products or biologics, which notice is for human administration. Reflects approval by the manufacturer, use, or distributor for this purpose.

Further provided is a kit that can be used in the manner described above. In one embodiment, the kit comprises the antibodies disclosed herein, preferably purified antibodies, in one or more containers. In certain embodiments, the kits disclosed herein contain a substantially isolated ApoC3 (eg, human ApoC3) antigen as a control. In another particular embodiment, the kit disclosed herein further comprises a control antibody that does not react with ApoC3 (eg, human ApoC3). In another particular embodiment, the kits disclosed herein contain one or more elements to detect binding of an antibody to an ApoC3 (eg, human ApoC3) antigen (eg, an antibody. A second antibody that recognizes a first antibody can be conjugated to a detectable substrate, such as a fluorescent compound, an enzyme substrate, a radioactive compound, or a luminescent compound). In certain embodiments, the kits provided herein can include recombinantly produced or chemically synthesized ApoC3 (eg, human ApoC3) antigens. The ApoC3 (eg, human ApoC3) antigen provided in the kit may also be attached to a solid support. In a more specific embodiment, the detection means of the above kit comprises a solid support to which the ApoC3 (eg, human ApoC3) antigen is bound. Such kits may also include unbound reporter-labeled anti-human or anti-mouse / rat antibodies. In this embodiment, the binding of the antibody to the ApoC3 (eg, human ApoC3) antigen can be detected by the binding of the reporter-labeled antibody.

The previously identified antibody clone 5E5 binds to ApoC3 with a high affinity at pH 7.4 and a slightly reduced affinity at pH 5.5 (see US Provisional Patent Application No. 62 / 360,084). sea bream). The present disclosure provides a novel derivative of clone 5E5 that exhibits high affinity binding to ApoC3 at pH 7.4 as compared to 5E5, but exhibits a lesser affinity for ApoC3 at pH 5.5. The following examples describe the characterization of the novel 5E5 derivative. The amino acid sequence of 5E5 is described in US Provisional Patent Application No. 62 / 360,084, and the amino acid sequence of the novel 5E5 derivative is shown in Tables 1-7 of the present specification.

The examples in this section are provided to further clarify the advantages and features of this application, but are not intended to limit the scope of this application. The examples are for illustrative purposes only.

Example 1: In vitro characterization of anti-ApoC3 scFv-Fc antibody.
This example describes a surface plasmon resonance (SPR) -based experiment to determine antigen-binding rates of anti-ApoC3 scFv-Fc antibodies at both pH 7.4 and pH 5.5.

A panel of novel derivatives of antibody clone 5E5 was generated by substitution of one or more CDR amino acids with histidine in VH and / or VL of 5E5. Antigen-binding rates of each 5E5 derivative at both pH 7.4 and pH 5.5 were evaluated using the SPR-based method and compared to 5E5 for high affinity binding to ApoC3 at pH 7.4. As shown, clones showing a lower affinity for ApoC3 at pH 5.5 were selected for further characterization. The binding rates of the exemplary 5E5 derivatives 5E5VH5_VLWT, 5E5VH12_VLWT, and 5E5VHWT_VL8 are shown in Table 14.

Test antibodies were produced from transfected HEK293 cells in 50 ml small cultures and purified by protein A chromatography using the AKTA pure chromatography system. The quality and yield of the purified antibody fragments were determined by spectrophotometry and SDS-PAGE.

Using the SPR-based method, biotinylated human ApoC3 was captured on a chip coated with streptavidin (SA) and the binding rate of the test antibody to the coated chip was measured at both pH 7.4 and pH 5.5. Briefly, 20 μl of biotinylated human ApoC3 was injected at a concentration of 10 μg / ml to reach a surface density of about 500 RU. 60 μl of each test antibody was added to HBS-EP buffer (GE, Catalog No. BR-1008-26; 0.010 M HEPES, 0.150 M NaCl, 3 mM EDTA, 0.05 (v / v)% detergent. Agent P20, pH 7.4) was diluted and injected at a concentration of 1-100 nM. The test antibody was passed through a flow cell at a flow rate of 30 μl / min followed by off-speed washing at pH 7.4 or pH 5.5 for 5 minutes. The obtained sensorgrams were analyzed using the BIA evolution 4.1 software to which the Langmuir 1: 1 binding model was applied to obtain the binding rate. The data was zero-adjusted and the reference cell sensorgram was decremented.

All scFv-Fc antibodies tested showed higher affinity for ApoC3 at pH 7.4 than pH 5.5, and antibody 5E5VH12_VLWT showed the most prominent pH-dependent binding (see Table 14). I want to be). The magnitude of the pH-dependent bond was positively correlated with the dissociation rate under acidic conditions.

Example 2: In vitro characterization of anti-ApoC3 human IgG ₁ antibody.
Based on the results of Example 1, the test scFv-Fc antibody was generated as a human IgG ₁ antibody. Using an SPR-based assay, human ApoC3 protein was immobilized on a CM5 chip and the binding rate of the test antibody to the coated chip was measured at both pH 7.4 and pH 5.5. Briefly, a solution of 50 μg / ml of natural human ApoC3 was prepared in 10 mM acetate buffer (pH 4.5) and injected until the surface density reached about 500 RU. 60 μl of each test antibody was added to HBS-EP buffer (GE, Catalog No. BR-1008-26, 0.010 M HEPES, 0.150 M NaCl, 3 mM EDTA, 0.05 (v / v)% surfactant. Agent P20, pH 7.4) was diluted and injected at the concentrations listed in Table 9. The test antibody was passed through a flow cell at a flow rate of 30 μl / min followed by off-speed washing at pH 7.4 or pH 5.5 for 5 minutes. The obtained sensorgrams were analyzed using the BIA evolution 4.1 software to which the Langmuir 1: 1 binding model was applied to obtain the binding rate. The data was zero-adjusted and the reference cell sensorgram was decremented.

All antibodies tested bound to human ApoC3 at pH 7.4 and had a reduced affinity for ApoC3 at pH 5.5 (see Table 15). 5E5VH5_VLWT, 5E5VH12_VLWT, 5E5VH5_VL8, and 5E5VH12_VL8 show partially significant pH-dependent binding.

Example 3: Effect of anti-ApoC3 antibody on uptake of VLDL by hepatocytes.
In this example, the ability of the anti-ApoC3 antibody to attenuate the uptake of VLDL by hepatocytes was determined.

Briefly, HepG2 cells (ATCC Hb-8065) were completely supplemented with 10% FCS-supplemented Complete Minimum Essential Medium (MEM) for 24 hours and 0.0125% bovine serum albumin (MEM-BSA medium). Cultured in MEM for an additional 24 hours on a poly-d-lysine coated surface. Cells were preincubated in fresh MEM-BSA medium with 3 μM human ApoC3 protein (Athens Research and Technology) and IgG ₁ format 3 μB test antibody for 15 minutes and 30 μg / mL ApoC3 depleted DiI labeled VLDL ( Kalen Biomedical, LLC # 770130-9) was added to the medium. After 4 hours of incubation, cells were further incubated for 20 minutes in a fresh complete MEM supplemented with 1% intralipide. The amount of DiI-labeled VLDL taken up by the cells was measured by lysing the cells with isopropanol for 15 minutes at room temperature, measuring the fluorescence of the DiI-labeled in the lysate (ex = 520 nm, em = 580) and using a standard curve. The amount of DiI labeled VLDL was calculated and determined by normalizing the data based on the total amount of protein in the lysate. The data are graphed using GraphPad Prism 6 and reported as mean +/- standard error. A one-way ANOVA with multiple comparisons was calculated using GraphPad Prism 6.

As shown in FIGS. 1A, 1B, and 1C, 5E5WT, 5E5VHWT_VL8, 5E5VH5_VLWT, 5E5VH12_VLWT, and 5E5VH5_VL8 antibodies increased VLDL uptake by HepG2 cells. In particular, the 5E5VHWT_VL8, 5E5VH5_VLWT, 5E5VH12_VLWT, and 5E5VH5_VL8 antibodies all fully restored VLDL uptake in the presence of ApoC3.

Example 4: Pharmacokinetics and Pharmacodynamics of Anti-ApoC3 Antibodies.
This example describes an in vivo characterization of the 5E5VH5_VL8 antibody using a mouse model in which triglyceride clearance does not function normally due to transgenic expression of human ApoC3.

4.1 Generation of mouse models Humans operably linked to the thyroxine-binding globulin (TBG) promoter (RegenXBio) in 60-63 day-old wild-type C57BL / 6 male mice maintained on a standard diet. 3 × 10 ¹¹ virus particles of the AAV8 vector carrying the ApoC3 gene were infected by intraperitoneal administration. Twelve days after dosing, blood samples were collected from the posterior orbital sinus and the levels of human ApoC3 in the blood samples were measured with the primary anti-ApoC3 antibody (Abcam rabbit polyclonal anti-human ApoC3 # ab21032) and the secondary ApoC3 antibody (Abcam goat polyclonal). Bioconjugated ApoC3 # ab21024) was used and measured by ELISA. In infected mice, the average serum level of human ApoC3 was 9.9 μΜ. The mean circulating triglyceride level after 4 hours of fasting was 163 mg / dL in these mice, while the mean circulating triglyceride level in the control mice was 109 mg / dL (p = 0.0065).

Mice were then grouped so that all groups had similar mean ApoC3 levels on day 12. Blood samples were collected from the posterior orbital sinus 14 days after AAV infection to establish baseline (T = 0) ApoC3 levels. A 25 mg / kg test anti-ApoC3 human IgG1 antibody was administered to _each mouse by injection into the dorsal subcutaneous cavity. Anti-chicken egg lysosomal human IgG ₁ antibody (HyHEL5) was used as an isotype control. Blood samples were collected from the posterior orbital sinus at 0, 2, 4, 8, and 24 hours after administration of the test antibody, and approximately every 2 days for the subsequent 30 days. All animal studies were performed according to the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. All procedures were approved by the Instrumental Animal Care and Use Committee of Vascamab, LLC.

4.2 Pharmacokinetics of Anti-ApoC3 Antibodies Mice expressing human ApoC3 were generated and treated as described in Section 4.1. Plasma levels of human IgG ₁ antibody were determined using an ELISA assay. Specifically, 96-well plates (Greener # 655061) were coated overnight at 4 ° C. with 50 μL of primary IgG antibody (Fitzgerald 41-XG57 goat anti-human IgG Fc polyclonal) diluted in PBS. Plates were washed 4 times with 200 μL TBS-T and 200 μL consisting of 3% BSA (Roche BSA fraction V protease free # 03 117 332 001) and clear milk (Pierce Clear Milk Blocker # 37587) in PBS. Blocked at 30 ° C. for 90 minutes with block buffer. Block buffer was removed, 50 μL of test sample diluted in block buffer was added and incubated for 2 hours at room temperature with rotation at 300 rpm. The plate was washed 4 times with 200 μL TBS-T, 50 μL of secondary antibody diluted in block buffer (Abcam goat anti-human IgG-Fc (biotin) polyclonal # ab97223) was added, and room temperature was rotated at 300 rpm. Was incubated for 1 hour. Plates were washed once with TBS-T, 50 μL of SA-HRP (Abcam # 64269) diluted 100-fold in PBS was added and incubated for 30 minutes at room temperature with rotation at 300 rpm. The plate was then washed 4 times with 200 μL TBS-T and developed with 80 μL TMB. The color reaction was stopped by 50 μL of 0.5 N HCL. Absorbance was read at a wavelength of 450 nm. The amount of human IgG in the test well was calculated from the four-parameter fit of the standard curves constructed using the purified test antibody. This method specifically detects human ApoC3 and does not cross-react with mouse ApoC3.

As shown in FIG. 2A, the 5E5 antibody was rapidly degraded in mice expressing human ApoC3. This can be explained by the rapid alternation of ApoC3 through the uptake of ApoC3-containing lipid particles. The 5E5VH5_VL8 antibody, which has a reduced affinity for ApoC3 at lower pH, can dissociate from ApoC3 in acidic organelles and return to the bloodstream via endosome reuse. The half-life of 5E5VH5_VL8 is about 1 week, which is similar to the half-life of the isotype control antibody HyHel5 (an antibody that does not bind to a specific antigen in mice). Plasma levels of 5E5VH5_VL8 return to baseline approximately 1 month after injection. The extended half-life of 5E5VH5_VL8 makes this antibody an excellent candidate for clinical use because of the low dosing frequency required to maintain therapeutic levels of the antibody in serum.

4.3 Pharmacodynamics of Anti-ApoC3 Antibodies Mice expressing human ApoC3 were generated and treated as described in Section 4.1. Plasma levels of human ApoC3 and ApoB were determined using an ELISA assay. Specifically, a 96-well plate (Greener # 655061) diluted with PBS is 50 μL of primary ApoC3 antibody (Abcam rabbit polyclonal anti-human ApoC3 # ab21032) or 50 μL of primary ApoB antibody (Meridian Life Sciences goat polyclonal anti-human ApoB #). K45253G) was coated overnight at 4 ° C. Plates were washed 4 times with 200 μL TBS-T and blocked with 200 μL block buffer (Pierce Clear Milk Blocker # 37587 in PBS) at 30 ° C. for 90 minutes. Block buffer was removed, 50 μL of test sample diluted with block buffer was added and incubated for 2 hours at room temperature with rotation at 300 rpm. Wash the plate 4 times with 200 μL TBS-T and dilute with block buffer 50 μL secondary ApoC3 antibody (Abcam goat polyclonal biotin conjugate ApoC3 # ab21024) or secondary ApoB antibody (Meridian Life Sciences goat polyclonal biotin conjugate). ApoB48 / 100 # 34003G) was added and incubated for 1 hour at room temperature with rotation at 300 rpm. Plates were washed once with TBS-T, 50 μL of SA-HRP (Abcam # 64269) diluted 100-fold in PBS was added and incubated for 30 minutes at room temperature with rotation at 300 rpm. The plate was then washed 4 times with 200 μL TBS-T and developed with 80 μL TMB (Thermo Ultra-TMB ELISA # 34028) followed by 50 μL 0.5N HCL. Absorbance was read at 450 nm. The amount of ApoC3 in the test well was calculated from the four-parameter conformance of the standard curve (Molecular Devices) constructed using purified ApoC3 (Athens Research and Technology). The amount of ApoB in the test wells was calculated from the four parameter fits of the standard curve (Molecular Devices) constructed using mouse VLDL isolated by centrifugation (ApoB content is assumed to be 20% of total protein content). do). Data were calculated and plotted as percentage values compared to the corresponding levels in mice treated with HyHel5 control antibody.

As shown in FIGS. 2B and 2C, the 5E5 antibody initially reduced plasma levels in human ApoC3 and ApoB, but the levels returned to normal after about 2 days. In contrast, 5E5VH5_VL8 reduced plasma levels of human ApoC3 and ApoB for about 1 month. This long shelf life coincided with the long half-life of 5E5VH5_VL8, confirming that 5E5VH5_VL8 would be an excellent clinical candidate.

Example 5: Humanization of anti-ApoC3 antibody.
Humanized variants of 5E5VH5_VL8 were generated according to the "germline" method described in WO20121235886A1. Briefly, human germline gene segments with the same standard folding structure and the highest amino acid sequence identity to the VH and VL regions of 5E5VH5_VL8 were identified by comparison with known human germline gene sequences. The closest human heavy and light chain germline sequences were human IGHV3-23 * 01 and human IGKV2-28 * 01, respectively. A phage display Fab library of the 5E5VH5_VL8 variant was constructed, with humanized mutations integrated into the amino acid residues of 5E5VH5_VL8, which differ from the human IGHV3-23 * 01 and IGKV2-28 * 01 germline sequences, and the llama residues and llama residues at each target location. Human residues were presented in the library as well. Mutations that remove the sequence-responsible motif DG, DS, NR, or M have also been incorporated into the library. After several rounds of affinity-driven phage display selection, the highest target binding properties (eg, high affinity binding to ApoC3 at pH 7.4, but less affinity for ApoC3 at pH 5.5). ) Has been identified as a humanized 5E5VH5_VL8 variant.

From the above initial selection and screening, 20 humanized anti-ApoC3 antibodies with high affinity, pH-dependent binding were identified. The VH and Vκ coding sequences of these antibodies were cloned into an expression vector and produced in ExpiCHO-S cells as a full-length IgG _single molecule. Antibodies were then purified using the Hitrap Mab Select Pure column (GE, Catalog No. 11-0034-94) in the AKTA Pure system. The antibody was eluted at pH 3.0 with 1.0 ml 0.1 M citrate buffer and the eluted fraction was placed in a tube containing 0.1 ml Tris-HCl pH 9.0 for neutralization. Collected. Using the HiTrap desalting column of the AKTA Pure system, the antibody-containing fractions were pooled and desalted with 1 × phosphate buffered saline (PBS; NaCl 137 mM, KCl 3 mM, Na2HPO4 8 mM, KH2PO4 15 mM, pH 7.4). Salted. The protein concentration was determined by measuring the absorbance at 280 nm and correcting the attenuation coefficient as follows: (A280 nm-A340 nm) / ε (attenuation coefficient (g / L)). Purified samples were tested by SDS-PAGE under reducing and non-reducing conditions to ensure the correct size and purity of the samples.

Two different surface plasmon resonance (SPR) assays were performed to evaluate the pH-dependent association and dissociation parameters of humanized anti-ApoC3 antibodies. The methods and results of the two assays are shown below.

5.1 SPR analysis of humanized anti-ApoC3 antibodies that bind to immobilized ApoC3 Biotinylated natural human ApoC3 at pH 7.4 by infusion of 20 μl of 10 μg / ml biotinylated natural human ApoC3 according to the method provided by Biacore. Captured on a streptavidin-coated SPR chip (GE Healthcare, Catalog No. BR1000032), thereby reaching a surface density of approximately 500 RU. HBS-EP buffer (GE, catalog number BR-1008-26, 0.010 M HEPES, 0.150 M NaCl, 3 mM EDTA, 0.05 (v / v)% surfactant P20, pH 7.4) 60 μL of test antibody in the range of 1-50 nM diluted in was injected, passed through a flow cell at a flow rate of 30 μl / min, followed by off-speed washing at pH 7.4 for 5 minutes. After dissociation, the flow cell surface was regenerated by injecting 10 μl of 10 mM NaOH / 1M NaCl and 10 μl of 10 mM glycine (pH 1.5). The assay was repeated at pH 5.5 using the same general protocol. Here, the running and sample dilution buffer was replaced with HBS-EP buffer supplemented with citrate buffer to a final concentration of 10 mM to adjust the pH to 5.5.

BIA evolution 4.1 software using the Langmuir 1: 1 binding model was used to analyze the resulting sensorgrams to obtain binding rates. The data was zero-adjusted and the reference cell sensorgram was decremented. Sensorgrams were doubly referenced in the sample-free blank assay. If the off-velocity value is below the device detection limit kd: 1 × ^10-6 1 / s, the matched parameters were considered inapplicable.

Table 16 outlines the calculated kinetic parameters for the humanized antibodies produced. FIG. 3 shows an overlaid sensorgram of each humanized mAb produced at a uniform mAb concentration of 25 nM at two assay pH values.

As shown in FIG. 3, mAb1 to mAb6 showed very low off-velocities at both pH 7.4 and pH 5.5. Their apparent affinity at pH 5.5 was improved compared to 5E5VH5_VL8, and no pH-dependent target binding was observed. The mAb7 antibody has a high off-rate at pH 5.5 and exhibits clear pH-dependent target binding. However, the Rmax value of the binding phase at both pH values was higher than the value of 5E5VH5_VL8. Antibodies from mAb8 to mAb12 showed very low off-rates at pH 7.4 and higher Rmax levels at pH 5.5 than pH 7.4. Their apparent affinity at pH 5.5 was improved compared to 5E5VH5_VL8, and no pH-dependent target binding was observed. The mAb13 and mAb14 antibodies were generated by transplanting the HCDR and LCDR sequences into the nearest human germline framework sequence and exhibited distinct pH-dependent target binding properties. However, their affinity for human ApoC3 was reduced at pH 7.4 compared to 5E5VH5_VL8. The mAb15, mAb17, and mAb19 antibodies did not contain histidine residues in LCDR3 (Q90H), but presented the desired pH-dependent target binding properties on the screen. The sequences of the mAb16, mAb18, and mAb20 antibodies were identical to mAb15, mAb17, and mAb19, respectively, except that the residue corresponding to H90 5E5VH5_VL8 was histidine. As shown in FIG. 3, mAb16, mAb18, and mAb20 showed clear pH-dependent target binding because of their reduced affinity for the target at pH 5.5 and increased off-rate at pH 7.4.

5.2 SPR analysis of ApoC3 binding to immobilized humanized anti-ApoC3 antibody A reverse SPR assay was designed to capture the test antibody on an SPR chip, inject human ApoC3 protein, and measure the binding of ApoC3 to the antibody. .. Specifically, a goat anti-human IgG Fcγ-specific antibody (Jackson ImmunoResearch, Catalog No. 109-005-098) was immobilized on a CM5 chip surface (GE Healthcare, Catalog No. BR100012). Immobilization was performed in 10 mM acetate buffer at pH 5.0 after activation of the chip according to the method provided by Biacore / GE using the NHS / EDC kit (Biacore AB, Catalog No. BR-1000-50). A solution of 30 μg / ml of anti-human Fcγ antibody was prepared and injected until the surface density reached about 10,000 RU. The analysis cycle consisted of the following steps:
(1) Antibody capture: HBS-EP buffer (GE, catalog number BR-1008-26; 0.010 M HEPES, 0.150 M NaCl, 3 mM EDTA, 0.05 (v / v)% surfactant The antibody is injected at a concentration of 50 nM at pH 7.4 or pH 5.5 diluted with activator P20, pH 7.4), allowing capture by high affinity anti-huFcγ antibody up to 800 RU.
(2) Baseline stabilization: Inject 100 μl of HBS-EP buffer at a flow rate of 30 μl / ml.
(3) Target binding: Inject 60 μl of the target ApoC3 protein diluted with HBS-EP buffer at concentrations of 400 nM, 200 nM, 100 nM, and 50 nM.
(4) Off-speed washing: Inject HBS-EP buffer at a flow rate of 30 μl / ml for 5 minutes to evaluate the dissociated phase.
(5) Chip regeneration by injecting 20 μl of 10 mM glycine at pH 1.5.

BIA evolution 4.1 software using the Langmuir 1: 1 binding model was used to analyze the resulting sensorgrams to obtain binding rates. The data was zero-adjusted and the reference cell sensorgram was decremented. A sample-free blank assay was used to double-reference the sensorgrams corresponding to the sample infusion. Dissociation and association phase sensorgrams were individually fitted to four different concentration curves. If the maximum RU value reached was below the detection limit of the device (<5RU), Biacore 3000 (Biacore AB), the sensorgram was excluded from the fitting.

Table 17 summarizes the calculated kinetic parameters for the humanized antibodies produced. FIG. 4 shows the binding properties of the generated variants. Due to the different presentation of target proteins, this setup increased the off rate of all antibodies.

As shown in FIG. 4, mAb1 to mAb6 showed an improved apparent affinity for ApoC3 at pH 7.4, indicating that pH-dependent target binding was observed. The mAb7 antibody had a high off-rate at both pH values and showed clear pH-dependent target binding. The Rmax value at pH 5.5 during the association period was increased compared to 5E5VH5_VL8. Antibodies from mAb8 to mAb12 show lower off-speeds at pH 7.4 than 5E5VH5_VL8, higher Rmax levels at pH 5.5 than pH 7.4, and higher at pH 5.5 than the off-speeds of 5E5VH5_VL8. It showed off-rate and showed pH-dependent target binding. The mAb13 and mAb14 antibodies (produced by transplanting the HCDR and LCDR sequences into the nearest human germline framework sequence) showed loss of binding at both pH levels and thus have an affinity value at pH 5.5. Was not calculated. The mAb16, mAb18, and mAb20 antibodies showed reduced affinity for the target at pH 5.5 and showed clear pH-dependent target binding, but also increased their off-rate at pH 7.4. rice field.

In summary, the following binding profile groups were identified for humanized antibodies according to the results of the SPR assay.
Group 1: mAb1, 2, 3, 4, 5, 6, 8, 9, 10, 11, and 12,
Group 2: mAb15, 17, and 19,
Group 3: mAb16, 18, and 20,
Group 4 comprises mAb7 and Group 5: mAb13 and 14.

Within each group, all variants showed similar SPR association and dissociation profiles. Therefore, representative antibodies from each group were selected and their thermostability was evaluated as one of their development potential parameters based on the highest human homology percentage criteria.

5.3 Thermostability characterization of selected humanized anti-ApoC3 antibodies The thermostability of humanized antibodies was assessed after incubation at different temperatures in the range 56-68 ° C. for 1 hour. The activity of the antibody was determined by an SPR assay in which 500 RU of native huApoC3 protein was immobilized on a CM5 chip (GE Healthcare, Catalog No. BR100012). Immobilization was performed using the NHS / EDC kit (Biacore AB) according to the method provided by Biacore, and after activation of the chip, 60 μg / ml human ApoC3 in 10 mM acetate buffer (pH 4.5). Solution was prepared and injected until the surface density reached about 500 RU.

HBS-EP buffer (GE, catalog number BR-1008-26, 0.010 M HEPES, 0.150 M NaCl, 3 mM EDTA, 0.05 (v / v)% surfactant P20, pH 7.4) 100 μL of the test antibody was injected at 10 nM diluted in the flow cell at a flow rate of 30 μl / min, followed by off-speed washing with HBS-EP buffer for 10 minutes at pH 7.4. After dissociation, the flow cell surface was regenerated by injecting 10 μl of 10 mM NaOH / 1M NaCl and 10 μl of 10 mM glycine (pH 1.5).

The association rate and the maximum RU value reached at the binding step were measured as read parameters. The association rate (gradient) was assessed at the initial binding stage up to 200 seconds and the RU maximum (R0) was read at the beginning of the dissociation stage at 345 seconds. The percentage of functional antibody was calculated based on a reference (sample incubated at 4 ° C) set to 100%. The melting temperature of each antibody was determined as an inflection in the activity vs. temperature curve.

As shown in FIGS. 5A and 5B, and as shown in Table 18, most humanized antibodies tested showed significantly improved melting temperatures compared to the parent clone 5E5VH5_VL8. However, mAb14 had a melting temperature significantly lower than 5E5VH5_VL8.

The thermostability of mAb13 was also evaluated in the assay described above to determine if the observed thermostability changes were associated with the germline framework sequences used in the CDR grafts of variants 13 and 14. The assay was performed over an extended temperature range to cover the expected temperature reached of the humanized antibody and 5E5VH5_VL8 (assayed as a control). As shown in FIG. 5C, mAb13 and mAb14 had similar heat resistance curves and showed a melting temperature of about 57 ° C., which was about 6 ° C. lower than the melting temperature of 5E5VH5_VL8.

The invention is not limited in scope by the particular embodiments disclosed herein. In fact, various modifications of the invention, in addition to those described, will be apparent to those skilled in the art from the above description and accompanying drawings. Such amendments are intended to be within the scope of the appended claims.

All references cited herein (eg, publications or patents or patent applications) are each individual reference (eg, publication or patent or patent application) specifically and individually for all purposes. To the same extent that the whole is shown to be incorporated by reference, the whole of them is incorporated herein by reference for all purposes.

Other embodiments are within the scope of the following claims.

Claims (22)

An isolated antibody that specifically binds to human ApoC3, wherein the antibody comprises heavy chain variable regions comprising complementarity determining regions CDRH1, CDRH2, and CDRH3, and complementarity determining regions CDRL1, CDRL2, and CDRL3. Contains light chain variable regions, and contains
(A) CDRH1 comprises the amino acid sequence of SEQ ID NO: 3.
(B) CDRH2 comprises the amino acid sequence of SEQ ID NO: 36.
(C) CDRH3 comprises the amino acid sequence of SEQ ID NO: 10.
(D) CDRL1 comprises the amino acid sequence of SEQ ID NO: 6.
(E) CDRL2 comprises the amino acid sequence of SEQ ID NO: 7, and (f) CDRL3 comprises the amino acid sequence of SEQ ID NO: 14.
Isolated antibody. The isolated antibody of claim 1, wherein the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 42. The isolated antibody of claim 1, wherein the light chain variable region comprises the amino acid sequence of SEQ ID NO: 54. The isolated antibody of claim 1, wherein the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 42 and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 54. The isolated antibody of claim 1, comprising a heavy chain comprising the amino acid sequence of SEQ ID NO: 68. The isolated antibody of claim 3, comprising a heavy chain comprising the amino acid sequence of SEQ ID NO: 68. The isolated antibody of claim 1, comprising a light chain comprising the amino acid sequence of SEQ ID NO: 74. The isolated antibody of claim 2, comprising a light chain comprising the amino acid sequence of SEQ ID NO: 74. An isolated antibody that specifically binds to human ApoC3, comprising a heavy chain comprising the amino acid sequence of SEQ ID NO: 68 and a light chain comprising the amino acid sequence of SEQ ID NO: 74. The isolated antibody according to claim 9 , wherein the amino acid sequence of the heavy chain consists of the amino acid sequence of SEQ ID NO: 68, and the amino acid sequence of the light chain consists of the amino acid sequence of SEQ ID NO: 74. The isolated antibody according to claim 1, which comprises a heavy chain constant region. The isolated antibody of claim 11 , wherein the heavy chain constant region is a heavy chain constant region of human IgG ₁ , IgG ₂ , or IgG ₄ . 11. The isolated antibody of claim 11 , wherein the heavy chain constant region comprises amino acids K, F, and Y at EU positions 433, 434, and 436, respectively. 11. The isolated antibody of claim 11 , wherein the heavy chain constant region comprises the amino acids Y, T, and E at EU positions 252, 254, and 256, respectively. 11. The isolated antibody of claim 11 , wherein the heavy chain constant region comprises amino acids L and S at EU positions 428 and 434, respectively. The amino acid sequence in which the heavy chain constant region is selected from the group consisting of SEQ ID NOs: 21, 22, 23, 24, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85 and 86. The isolated antibody of claim 11 , comprising. The isolated antibody according to claim 1, which comprises a light chain constant region. 17. The isolated antibody of claim 17 , wherein the light chain constant region comprises the amino acid sequence of SEQ ID NO: 25 or 26. A pharmaceutical composition comprising the antibody of claim 1 and a pharmaceutically acceptable carrier. A pharmaceutical composition comprising the antibody of claim 4 and a pharmaceutically acceptable carrier. A pharmaceutical composition comprising the antibody of claim 9 and a pharmaceutically acceptable carrier. A pharmaceutical composition comprising the antibody of claim 10 and a pharmaceutically acceptable carrier.

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